Anti-abeta globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies

ABSTRACT

Anti-Aβ globulomer antibodies, antigen-binding moieties thereof, corresponding hybridomas, nucleic acids, vectors, host cells, methods of producing said antibodies, compositions comprising said antibodies, uses of said antibodies and methods of using said antibodies. 
     The present invention relates to anti-Aβ globulomer antibodies having a binding affinity to Aβ(20-42) globulomer that is greater than the binding affinity of the antibody to Aβ(1-42) globulomer, antigen-binding moieties thereof, hybridomas producing said antibodies, nucleic acids encoding said antibodies, vectors comprising said nucleic acids, host cells comprising said vectors, methods of producing said antibodies, compositions comprising said antibodies, therapeutic and diagnostic uses of said antibodies and corresponding methods relating to Alzheimer&#39;s disease and other amyloidoses.

The present invention relates to anti-Aβ globulomer antibodies,antigen-binding moieties thereof, hybridomas producing said antibodies,nucleic acids encoding said antibodies, vectors comprising said nucleicacids, host cells comprising said vectors, methods of producing saidantibodies, compositions comprising said antibodies, therapeutic anddiagnostic uses of said antibodies and corresponding methods relating toAlzheimer's disease and other amyloidoses.

In 1907 the physician Alois Alzheimer first described theneuropathological features of a form of dementia subsequently named inhis honour (Alzheimer 1907). Alzheimer's disease (AD) is the mostfrequent cause for dementia among the aged with an incidence of about10% of the population above 65 years. With increasing age, theprobability of disease also rises. Globally, there are about 15 millionpeople affected and further increases in life expectancy are expected toincrease the number of diseased people to about threefold over the nextdecades.

From a molecular point of view Alzheimer's disease (AD) is characterizedby a deposit of abnormally aggregated proteins. In the case ofextra-cellular amyloid plaques these deposits consist mostly ofamyloid-β-peptide filaments, in the case of the intracellularneurofibrillary tangles (NFTs) of the tau protein. The amyloid β (Aβ)peptide arises from the β-amyloid precursor protein by proteolyticcleavage. This cleavage is effected by the cooperative activity ofseveral proteases named α-, β- and γ-secretase. Cleavage leads to anumber of specific fragments of differing length. The amyloid plaquesconsist mostly of peptides with a length of 40 or 42 amino acids (Aβ40,Aβ342) The dominant cleavage product is Aβ40; however, Aβ42 has a muchstronger toxic effect.

Cerebral amyloid deposits and cognitive impairments very similar tothose observed in Alzheimer's disease are also hallmarks of Down'ssyndrome (trisomy 21), which occurs at a frequency of about 1 In 800births.

The amyloid cascade hypothesis of Hardy and Higgins postulated thatincreased production of Aβ(1-42) would lead to the formation ofprotofibrils and fibrils, the principal components of Aβ plaques, thesefibrils being responsible for the symptoms of Alzheimer's disease.Despite the poor correlation between severity of dementia and Aβ plaqueburden deposited this hypothesis was favoured until recently. Thediscovery of soluble Aβ forms in AD brains, which correlates better withAD symptoms than plaque load does, has led to a revisedamyloid-cascade-hypothesis.

Active immunization with Aβ peptides leads to a reduction in theformation as well as to partial dissolution of existing plaques. At thesame time it leads to alleviation of cognitive defects in APP transgenicmouse models.

For passive immunization with antibodies directed to Aβ peptides areduction of an Aβ plaque burden was also found.

The results of a phase IIa trial (ELAN Corporation Plc, South SanFrancisco, Calif., USA and Dublin, UK) of active immunization withAN-1792 (Aβ(1-42) peptide in fibrillary condition of aggregation)suggest that immunotherapy directed to Aβ peptide was successful. In asubgroup of 30 patients the progression of disease was significantlyreduced in patients with positive anti-Aβ antibody titer, measured byMMSE and DAD index. However, this study was stopped because of seriousside effects in form of a meningoencephalitis (Bennett and Holtzman,2005, Neurology, 64, 10-12).

Meningoencephalitis was characterized by neuroinflammation andinfiltration of T-cells into the brain. Presumably, this was due to aT-cell immune response induced by injection of Aβ(1-42) as antigen. Suchan immune response is not to be expected after passive immunization. Todate, there are no clinical data with reference to this available yet.However, with reference to such a passive approach to immunizationconcerns about the side effect profile were voiced because ofpreclinical studies in very old APP23 mice which received an antibodydirected against an N-terminal epitope of Aβ(1-42) once a week over 5months. These mice showed an increase in the number and severity ofmicrohaemorrhages compared to control animals treated with saline(Pfeffer et al., 2002, Science, 298, 1379). A comparable increase inmicrohaemorrhages was also described in very old (>24 months) Tg2576 andPDAPP mice (Racke et al., 2005, J Neuroscl, 25, 629-636; Wilcock et al.2004, J. Neuroinflammation, 1(1):24; De Mattos et al., 2004, Neurobiol.Aging 25(S2):577). In both mouse strains antibody injection led to asignificant increase in microhaemorrhages. In contrast, an antibodydirected against the central region of the Aβ(1-42) peptide did notinduce microhaemorrhages (de Mattos et al., supra). The lack of inducingmicrohaemorrhages was associated with an antibody treatment which didnot bind to aggregated Aβ peptide in the form of CAA (Racke et al., JNeuroscl, 25, 629-636). But, the exact mechanism leading tomicrohaemorrhages in mice transgenic for APP has not been understood.Presumably, cerebral amyoid angiopathy (CAA) induces or at leastaggravates cerebral haemorrhages. CAA is present in nearly everyAlzheimer's disease brain and about 20% of the cases are regarded as“severe CAA”. Passive immunization should therefore aim at avoidingmicrohaemorrhages by selecting an antibody which recognizes the centralor the carboxy terminal region of the Aβ peptide.

WO2004/067561 describes stable Aβ(1-42) oligomers (Aβ(1-42) globulomers)and antibodies directed specifically against the globulomers. Digestionwith unspecific proteases shows that the Aβ globulomer may be digestedbeginning with the hydrophilic N-terminus protruding from the globularcore structure (Barghorn et al., 2005, J Neurochem, 95, 834-847). SuchN-terminal truncated Aβ globulomers (Aβ(12-42) and Aβ(20-42)globulomers) represent the basic structural unit of this oligomeric AβThey are a very potent antigen for active immunization of rabbits andmice leading to high antibody titers (WO2004/067561). The putativepathological role of N-terminally truncated Aβ forms in vivo has beensuggested by several recent reports of their existence in AD brains(Sergeant at al., 2003, J Neurochem, 85, 1581-1591; Thal et al., 1999, JNeuropathol. Exp Neurol, 58, 210-216). During in vivo digestion certainproteases found in brain, e.g. neprilysin (NEP 24.11) or insulindegrading enzyme (IDE), may be involved (Selkoe, 2001, Neuron, 32,177-180).

It was an object of the present invention to provide antibodies directedagainst A, globulomers which improve the cognitive performance of apatient in immunotherapy while at the same time reacting only with asmall portion of the entire amount of Aβ peptide in brain. This isexpected to prevent a substantial disturbance of cerebral Aβ balance andlead to less side effects. (For instance, a therapeutically questionablereduction of brain volume has been observed in the study of activeimmunization with Aβ peptides in fibrillary condition of aggregation(ELAN trial with AN1792). Moreover, in this trial severe side effects inform of a meningoencephalitis were observed.

The present invention solves this problem by providingglobulomer-specific antibodies possessing high affinity for truncatedforms of AB globulomers. These antibodies are capable of discriminatingnot only other forms of Aβ peptides, particularly monomers and flbrils,but also untruncated forms of Aβ globulomers.

Thus, the present invention relates to an antibody having a bindingaffinity to an Aβ(20-42) globulomer that is greater than the bindingaffinity of this antibody to an Aβ(1-42) globulomer.

Further, the present invention relates to an antibody having a bindingaffinity to an Aβ(20-42) globulomer that is greater than the bindingaffinity of this antibody to an Aβ(12-42) globulomer.

According to a particular embodiment, the invention thus relates toantibodies having a binding affinity to the Aβ(20-42) globulomer that isgreater than the binding affinity of the antibody to both the Aβ(1-42)globulomer and the Aβ(12-42) globulomer.

The term “Aβ(X-Y)” here refers to the amino acid sequence from aminoacid position X to amino acid position Y of the human amyloid 1 proteinincluding both X and Y, in particular to the amino acid sequence fromamino acid position X to amino acid position Y of the amino acidsequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGW IAT (correspondingto amino acid positions 1 to 43) or any of its naturally occurringvariants, in particular those with at least one mutation selected fromthe group consisting of A2T, H6R, D7N, A21G (“Flemish”), E22G(“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T andA42V wherein the numbers are relative to the start of the Aβ peptide,including both position X and position Y or a sequence with up to threeadditional amino acid substitutions none of which may prevent globulomerformation, preferably with no additional amino acid substitutions in theportion from amino acid 12 or X, whichever number is higher, to aminoacid 42 or Y, whichever number is lower, more preferably with noadditional amino acid substitutions in the portion from amino acid 20 orX, whichever number is higher, to amino acid 42 or Y, whichever numberIs lower, and most preferably with no additional amino acidsubstitutions in the portion from amino acid 20 or X, whichever numberis higher, to amino acid 40 or Y, whichever number is lower, an“additional” amino acid substation herein being any deviation from thecanonical sequence that is not found in nature.

More specifically, the term “Aβ(1-42)” here refers to the amino acidsequence from amino acid position 1 to amino acid position 42 of thehuman amyloid β protein including both 1 and 42, in particular to theamino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGW IA or anyof its naturally occurring variants, in particular those with at leastone mutation selected from the group consisting of A2T, H6R, D7N, A21G(“Flemish”), E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N(“Iowa”), A42T and A42V wherein the numbers are relative to the start ofthe Aβ peptide, including both 1 and 42 or a sequence with up to threeadditional amino acid substitutions none of which may prevent globulomerformation, preferably with no additional amino acid substitutions in theportion from amino acid 20 to amino acid 42. Likewise, the term“Aβ(1-40)” here refers to the amino acid sequence from amino acidposition 1 to amino acid position 40 of the human amyloid β proteinincluding both 1 and 40, in particular to the amino acid sequenceDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGW or any of Its naturallyoccurring variants, in particular those with at least one mutationselected from the group consisting of A2T, H6R, D7N, A21G (“Flemish”),E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”), and D23N (“Iowa”)wherein the numbers are relative to the start of the Aβ peptide,including both 1 and 40 or a sequence with up to three additional aminoacid substitutions none of which may prevent globulomer formation,preferably with no additional amino acid substitutions in the portionfrom amino acid 20 to amino acid 40.

More specifically, the term “Aβ(12-42)” here refers to the amino acidsequence from amino acid position 12 to amino acid position 42 of thehuman amyloid β protein including both 12 and 42, in particular to theamino acid sequence VHHQKLVFF AEDVGSNKGA IIGLMVGGW IA or any of itsnaturally occurring variants, in particular those with at least onemutation selected from the group consisting of A21G (“Flemish”), E22G(“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T andA42V wherein the numbers are relative to the start of the Aβ peptide,including both 12 and 42 or a sequence with up to three additional aminoacid substitutions none of which may prevent globulomer formation,preferably with no additional amino acid substitutions in the portionfrom amino acid 20 to amino acid 42.

More specifically, the term “Aβ(20-42)” here refers to the amino acidsequence from amino add position 20 to amino acid position 42 of thehuman amyloid β protein including both 20 and 42, in particular to theamino acid sequence F AEDVGSNKGA IIGLMVGGW IA or any of its naturallyoccurring variants, in particular those with at least one mutationselected from the group consisting of A21G (“Flemish”), E22G (“Arctic”),E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V whereinthe numbers are relative to the start of the Aβ peptide, including both20 and 42 or a sequence with up to three additional amino acidsubstitutions none of which may prevent globulomer formation, preferablywithout any additional amino acid substitutions.

The term “Aβ(X-Y) globulomer” (Aβ(X-Y) globular oligomer) here refers toa soluble, globular, non-covalent association of Aβ(X-Y) peptides asdefined above, possessing homogeneity and distinct physicalcharacteristics. According to one aspect, Aβ(X-Y) globulomers arestable, non-fibrillar, oligomeric assemblies of Aβ(X-Y) peptides whichare obtainable by incubation with anionic detergents. In contrast tomonomer and fibris, these globulomers are characterized by definedassembly numbers of subunits (e.g. early assembly forms, n=4-6,“oligomers A”, and late assembly forms, n=12-14, “oligomers B”, asdescribed in WO2004/067561). The globulomers have a 3-dimensionalglobular type structure (“molten globule”, see Barghorn et al., 2005, JNeurochem, 95, 834-847) They may be further characterized by one or moreof the following features:

-   -   cleavability of N-terminal amino acids X-23 with promiscuous        proteases (such as thermolysin or endoproteinase GluC) yielding        truncated forms of globulomers;    -   non-accessibility of C-terminal amino acids 24-Y with        promiscuous proteases and antibodies;    -   truncated forms of these globulomers maintain the 3-dimensional        core structure of said globulomers with a better accessibility        of the core epitope Aβ(20-Y) in its globulomer conformation.

According to the Invention and in particular for the purpose ofassessing the binding affinities of the antibodies of the presentinvention, the term “Aβ(X-Y) globulomer” here refers in particular to aproduct which is obtainable by a process as described in WO 2004/067561,which is incorporated herein by reference.

Said process comprises unfolding a natural, recombinant or syntheticAβ(X-Y) peptide or a derivative thereof, exposing the at least partiallyunfolded Aβ(X-Y) peptide or derivative thereof to a detergent, reducingthe detergent action and continuing incubation.

For the purpose of unfolding the peptide, hydrogen bond-breaking agentssuch as, for example, hexafluoroisopropanol (HFIP) may be allowed to acton the protein. Times of action of a few minutes, for example about 10to 60 minutes, are sufficient when the temperature of action is fromabout 20 to 50° C. and in particular about 35 to 40° C. Subsequentdissolution of the residue evaporated to dryness, preferably inconcentrated form, in suitable organic solvents miscible with aqueousbuffers, such as, for example, dimethyl sulfoxide (DMSO), results in asuspension of the at least partially unfolded peptide or derivativethereof, which can be used subsequently. If required, the stocksuspension may be stored at low temperature, for example at about −20°C., for an interim period.

Alternatively, the peptide or the derivative thereof may be taken up inslightly acidic, preferably aqueous, solution, for example an about 10mM aqueous HCl solution. After an incubation time of usually a fewminutes, insoluble components are removed by centrifugation. A fewminutes at 10000 g is expedient. These method steps are preferablycarried out at room temperature, i.e. a temperature in the range from 20to 30° C. The supernatant obtained after centrifugation contains theAβ(X-Y) peptide or the derivative thereof and may be stored at lowtemperature, for example at about −20° C., for an interim period.

The following exposure to a detergent relates to the oligomerization ofthe peptide or the derivative thereof to give an intermediate type ofoligomers (in WO 2004/067561 referred to as oligomers A). For thispurpose, a detergent is allowed to act on the at least partiallyunfolded peptide or derivative thereof until sufficient intermediateoligomer has been produced.

Preference is given to using ionic detergents, in particular anionicdetergents.

According to a particular embodiment, a detergent of the formula (I):

R—X,

is used, in whichthe radical R is unbranched or branched alkyl having from 6 to 20 andpreferably 10 to 14 carbon atoms or unbranched or branched alkenylhaving from 6 to 20 and preferably 10 to 14 carbon atoms,the radical X is an acidic group or salt thereof, with X beingpreferably selected from among —COO⁻M⁺, —SO₃ ⁻M⁺, and especially—OSO₃ ⁻M⁺ and M⁺ is a hydrogen cation or an inorganic or organic cationpreferably selected from alkali metal and alkaline earth metal cationsand ammonium cations.

Advantageous are detergents of the formula (I), in which R is unbranchedalkyl of which alk-1-yl radicals must be mentioned in particular.Particular preference is given to sodium dodecyl sulfate (SDS). Lauricacid and oleic acid can also be used advantageously. The sodium salt ofthe detergent lauroylsarcosin (also known as sarkosyl NL-30 or Gardol®)is also particularly advantageous.

The time of detergent action in particular depends on whether—and ifyes, to what extent—the peptide or the derivative thereof subjected tooligomerization has unfolded. If, according to the unfolding step, thepeptide or derivative thereof has been treated beforehand with ahydrogen bond-breaking agent, i.e. in particular withhexafluoroisopropanol, times of action in the range of a few hours,advantageously from about 1 to 20 and in particular from about 2 to 10hours, are sufficient when the temperature of action is about 20 to 50°C. and in particular about 35 to 40° C. If a less unfolded or anessentially not unfolded peptide or derivative thereof is the startingpoint, correspondingly longer times of action are expedient. If thepeptide or the derivative thereof has been pretreated, for example,according to the procedure indicated above as an alternative to the HFIPtreatment or said peptide or derivative thereof is directly subjected tooligomerization, times of action in the range from about 5 to 30 hoursand in particular from about 10 to 20 hours are sufficient when thetemperature of action is about 20 to 50° C. and in particular about 35to 40° C. After incubation, insoluble components are advantageouslyremoved by centrifugation. A few minutes at 10000 g is expedient.

The detergent concentration to be chosen depends on the detergent used.If SDS is used, a concentration in the range from 0.01 to 1% by weight,preferably from 0.05 to 0.5% by weight, for example of about 0.2% byweight, proves expedient. If lauric acid or oleic acid are used,somewhat higher concentrations are expedient, for example in a rangefrom 0.05 to 2% by weight, preferably from 0.1 to 0.5% by weight, forexample of about 0.5% by weight.

The detergent action should take place at a salt concentrationapproximately in the physiological range. Thus, in particular NaClconcentrations in the range from 50 to 500 mM, preferably from 100 to200 mM and particularly at about 140 mM are expedient.

The subsequent reduction of the detergent action and continuation ofincubation relates to a further oligomerization to give the Aβ(X-Y)globulomer of the invention (In WO 2004/067561 referred to as oligomersB). Since the composition obtained from the preceding step regularlycontains detergent and a salt concentration in the physiological rangeit is then expedient to reduce detergent action and, preferably, alsothe salt concentration. This may be carried out by reducing theconcentration of detergent and salt, for example, by diluting,expediently with water or a buffer of lower salt concentration, forexample Tris-HCl, pH 7.3. Dilution factors in the range from about 2 to10, advantageously in the range from about 3 to 8 and in particular ofabout 4, have proved suitable. The reduction in detergent action mayalso be achieved by adding substances which can neutralize saiddetergent action Examples of these include substances capable ofcomplexing the detergents, like substances capable of stabilizing cellsin the course of purification and extraction measures, for exampleparticular EO/PO block copolymers, in particular the block copolymerunder the trade name Pluronic® F 68. Alkoxylated and, in particular,ethoxylated alkyl phenols such as the ethoxylated t-octylphenols of theTriton° X series, in particular Triton® X100,3-(3-cholamidopropydimethylammonio)-1-propanesulfonate (CHAPS®) oralkoxylated and, in particular, ethoxylated sorbitan fatty esters suchas those of the Tween® series, in particular Tween® 20, in concentrationranges around or above the particular critical micelle concentration,may be equally used.

Subsequently, the solution is incubated until sufficient Aβ(X-Y)globulomer of the invention has been produced. Times of action in therange of several hours, preferably in the range from about 10 to 30hours and in particular in the range from about 15 to 25 hours, aresufficient when the temperature of action is about 20 to 50° C. and inparticular about 35 to 40° C. The solution may then be concentrated andpossible residues may be removed by centrifugation. Here too, a fewminutes at 10000 g proves expedient. The supernatant obtained aftercentrifugation contains an Aβ(X-Y) globulomer of the invention.

An Aβ(X-Y) globulomer of the invention can be finally recovered in amanner known per se, e. g. by ultrafiltration, dialysis, precipitationor centrifugation.

It is further preferred if electrophoretic separation of the Aβ(X-Y)globulomers under denaturing conditions, e. g. by SDS-PAGE, produces adouble band (e. g. with an apparent molecular weight of 38/48 kDa forAβ(1-42)), and especially preferred if upon glutardialdehyde treatmentof the globulomers before separation these two bands are merged intoone. It is also preferred if size exclusion chromatography of theglobulomers results in a single peak (e. g. corresponding to a molecularweight of approximately 100 kDa for Aβ(1-42) globulomer or ofapproximately 60 kDa for glutardialdehyde cross-linked Aβ(1-42)globulomer), respectively.

Starting out from Aβ(1-42) peptide, Aβ(12-42) peptide, and Aβ(20-42)peptide said processes are in particular suitable for obtaining Aβ(1-42)globulomers, Aβ(12-42) globulomers, and Aβ(20-42) globulomers.

In a particular embodiment of the invention, Aβ(X-Y) globulomers whereinX is selected from the group consisting of the numbers 2 . . . 24 and Yis as defined above, are those which are obtainable by truncatingAβ(1-Y) globulomers into shorter forms wherein X is selected from thegroup consisting of the numbers 2 . . . 24, with X preferably being 20or 12, and Y is as defined above, which can be achieved by treatmentwith appropriate proteases. For instance, an Aβ(20-42) globulomer can beobtained by subjecting an Aβ(1-42) globulomer to thermolysinproteolysis, and an Aβ(12-42) globulomer can be obtained by subjectingan Aβ(1-42) globulomer to endoproteinase GluC proteolysis. When thedesired degree of proteolysis is reached, the protease is inactivated ina generally known manner. The resulting globulomers may then be isolatedfollowing the procedures already described herein and, if required,processed further by further work-up and purification steps. A detaileddescription of said processes is disdosed in WO 2004/067561, which isincorporated herein by reference.

For the purposes of the present invention, an Aβ(1-42) globulomer is inparticular the Aβ(1-42) globulomer as described in example 1a herein; anAβ(20-42) globulomer is in particular the Aβ(20-42) globulomer asdescribed in examples 1c herein, and an Aβ(12-42) globulomer is inparticular the Aβ(12-42) globulomer as described in examples 1d herein.

Preferably, the globulomer shows affinity to neuronal cells. Preferably,the globulomer also exhibits neuromodulating effects.

According to another aspect of the invention, the globulomer consists of11 to 16, and most preferably, of 12 to 14 Aβ(X-Y) peptides.

According to another aspect of the invention, the term “Aβ(X-Y)globulomer” here refers to a globulomer consisting essentially ofAβ(X-Y) subunits, where it is preferred if on average at least 11 of 12subunits are of the Aβ(X-Y) type, more preferred if less than 10% of theglobulomers comprise any non-Aβ(X-Y) peptides, and most preferred if thecontent of non-Aβ(X-Y) peptides is below the detection threshold.

More specifically, the term “Aβ(1-42) globulomer” here refers to aglobulomer consisting essentially of Aβ(1-42) units as defined above;the term “Aβ(12-42) globulomer” here refers to a globulomer consistingessentially of Aβ(12-42) units as defined above; and the term “Aβ(20-42)globulomer” here refers to a globulomer consisting essentially ofAβ(20-42) units as defined above.

The term “cross-linked Aβ(X-Y) globulomer” here refers to a moleculeobtainable from an Aβ(X-Y) globulomer as described above bycross-linking, preferably chemically cross-linking, more preferablyaldehyde cross-linking, most preferably glutardialdehyde cross-linkingof the constituent units of the globulomer. In another aspect of theinvention, a cross-linked globulomer is essentially a globulomer inwhich the units are at least partially joined by covalent bonds, ratherthan being held together by non-covalent interactions only. For thepurposes of the present invention, a cross-linked Aβ(1-42) globulomer isin particular the cross-linked Aβ(1-42) oligomer as described in example1b herein.

The term “Aβ(X-Y) globulomer derivative” here refers in particular to aglobulomer that is labelled by being covalently linked to a group thatfacilitates detection, preferably a fluorophore, e. g. fluoresceinisothiocyanate, phycoerythrin, Aequorea victoria fluorescent protein,Dictyosoma fluorescent protein or any combination or fluorescence-activederivative thereof; a chromophore; a chemoluminophore, e. g. luciferase,preferably Photinus pyralis luciferase, Vibrio fischeri luciferase, orany combination or chemoluminescence-active derivative thereof; anenzymatically active group, e. g. peroxidase, e. g. horseradishperoxidase, or any enzymatically active derivative thereof; anelectron-dense group, e. g. a heavy metal containing group, e.g. a goldcontaining group; a hapten, e. g. a phenol derived hapten; a stronglyantigenic structure, e. g. peptide sequence predicted to be antigenic,e. g. predicted to be antigenic by the algorithm of Kolaskar andTongaonkar; an aptamer for another molecule; a chelating group, e. g.hexahistidinyl; a natural or nature-derived protein structure mediatingfurther specific protein-protein interactions, e. g. a member of thefos/jun pair; a magnetic group, e. g. a ferromagnetic group; or aradioactive group, e. g. a group comprising ¹H, ¹⁴C, ²P, ³S or ¹²⁵I orany combination thereof; or to a globulomer flagged by being covalentlyor by non-covalent high-affinity interaction, preferably covalentlylinked to a group that facilitates inactivation, sequestration,degradation and/or precipitation, preferably flagged with a group thatpromotes in vivo degradation, more preferably with ubiquitin, where isparticularly preferred if this flagged oligomer is assembled in vivo; orto a globulomer modified by any combination of the above. Such labellingand flagging groups and methods for attaching them to proteins are knownin the art. Labelling and/or flagging may be performed before, during orafter globulomerisation. In another aspect of the invention, aglobulomer derivative is a molecule obtainable from a globulomer by alabelling and/or flagging reaction.

Correspondingly, term “Aβ(X-Y) monomer derivative” here refers inparticular to an Aβ monomer that is labelled or flagged as described forthe globulomer.

Expediently, the antibody of the present invention binds to an Aβ(20-42)globulomer with a K_(D) in the range of 1×10⁻⁶ M to 1×10⁻¹² M.Preferably, the antibody binds to an Aβ(20-42) globulomer with highaffinity, for instance with a K_(D) of 1×10⁻⁷ M or greater affinity,e.g. with a K_(D) of 3×10⁻⁸ M or greater affinity, with a K_(D) of1×10⁻⁸ M or greater affinity, e.g. with a K_(D) of 3×10⁻⁹ M or greateraffinity, with a K_(D) of 1×10⁻⁹ M or greater affinity, e.g. with aK_(D) of 3×10⁻¹⁰ M or greater affinity, with a K_(D) of 1×10⁻¹⁰ M orgreater affinity, e.g. with a K_(D) of 3×10⁻¹¹ M or greater affinity, orwith a K_(D) of 1×10⁻¹¹ M or greater affinity.

The term “greater affinity” here refers to a degree of interaction wherethe equilibrium between unbound antibody and unbound globulomer on theone hand and antibody-globulomer complex on the other is further infavour of the antibody-globulomer complex. Likewise, the term “smalleraffinity” here refers to a degree of interaction where the equilibriumbetween unbound antibody and unbound globulomer on the one hand andantibody-globulomer complex on the other is further in favour of theunbound antibody and unbound globulomer. The term “greater affinity” issynonymous with the term “higher affinity” and term “smaller affinity”is synonymous with the term “lower affinity”.

According to a particular embodiment, the invention relates to anantibody which binds to the Aβ(20-42) globulomer with a K_(D) in therange of 1×10⁻⁶ M to 1×10⁻¹² M, to the Aβ(1-42) globulomer with a K_(D)of 10⁻¹² M or smaller affinity, the binding affinity to the Aβ(20-42)globulomer being greater than the binding affinity to the Aβ(1-42)globulomer.

It is preferred that the binding affinity of the antibody of the presentinvention to the Aβ(20-42) globulomer is at least 2 times, e. g. atleast 3 times or at least 5 times, preferably at least 10 times, e. g.at least 20 times, at least 30 times or at least 50 times, morepreferably at least 100 times, e. g. at least 200 times, at least 300times or at least 500 times, and even more preferably at least 1000times, e. g. at least 2000 times, at least 3000 times or at least 5000times, even more preferably at least 10000 times, e. g. at least 20000times, at least 30000 or at least 50000 times, and most preferably atleast 100000 times greater than the binding affinity of the antibody tothe Aβ(1-42) globulomer.

According to a particular embodiment, the invention relates to anantibody which binds to the Aβ(12-42) globulomer with a K_(D) with aK_(D) of 10⁻¹² M or smaller affinity, the binding affinity to theAβ(20-42) globulomer being greater than the binding affinity to theAβ(12-42) globulomer.

It is also preferred that the binding affinity of the antibody of thepresent invention to the Aβ(20-42) globulomer is at least 2 times, e. g.at least 3 times or at least 5 times, preferably at least 10 times, e.g. at least 20 times, at least 30 times or at least 50 times, morepreferably at least 100 times, e. g. at least 200 times, at least 300times or at least 500 times, and even more preferably at least 1000times, e. g. at least 2000 times, at least 3000 times or at least 5000times, even more preferably at least 10000 times, e. g. at least 20000times, at least 30000 or at least 50000 times, and most preferably atleast 100000 times greater than the binding affinity of the antibody tothe Aβ(12-42) globulomer.

Preferably, the antibodies of the present invention bind to at least oneAβ globulomer, as defined above, and have a comparatively smalleraffinity for at least one non-globulomer form of Aβ.

Antibodies of the present invention having a comparatively smalleraffinity for at least one non-globulomer form of Aβ than for at leastone Aβ globulomer include antibodies having a binding affinity to theAβ(20-42) globulomer that is greater than to an Aβ(1-42) monomer.Further, it is preferred that, alternatively or additionally, thebinding affinity of the antibody to the Aβ(20-42) globulomer is greaterthan to an Aβ(1-40) monomer.

In a preferred embodiment of the invention, the affinity of the antibodyto the Aβ(20-42) globulomer is greater than its affinity to both theAβ(1-40) and the Aβ(1-42) monomer.

The term “Aβ(X-Y) monomer” here refers to the isolated form of theAβ(X-Y) peptide, preferably a form of the Aβ(X-Y) peptide which is notengaged in essentially non-covalent interactions with other Aβ peptidesPractically, the Aβ(X-Y) monomer is usually provided in the form of anaqueous solution. In a particularly preferred embodiment of theinvention, the aqueous monomer solution contains 0.05% to 0.2%, morepreferably about 0.1% NH₄OH. In another particularly preferredembodiment of the invention, the aqueous monomer solution contains 0.05%to 0.2%, more preferably about 0.1% NaOH. When used (for instance fordetermining the binding affinities of the antibodies of the presentinvention), it may be expedient to dilute said solution in anappropriate manner. Further, it is usually expedient to use saidsolution within 2 hours, in particular within 1 hour, and especiallywithin 30 minutes after its preparation.

More specifically, the term “Aβ(1-40) monomer” here refers to anAβ(1-40) monomer preparation as described in example 2 herein, and theterm “Aβ(1-42) monomer” here refers to an Aβ(1-42) preparation asdescribed in example 2 herein.

Expediently, the antibody of the present invention binds to one or, morepreferably, both monomers with low affinity, most preferably with aK_(D) of 1×10 M or smaller affinity, e. g. with a K_(D) of 3×10⁴ M orsmaller affinity, with a K_(D) of 1×10⁻⁷ M or smaller affinity, e. g.with a K_(D) of 3×10⁻⁷ M or smaller affinity, or with a K_(D) of 1×10⁻⁷M or smaller affinity, e. g. with a K_(D) of 3×10⁻⁷ M or smalleraffinity, or with a K_(D) of 1×10⁻⁵ M or smaller affinity.

It is especially preferred that the binding affinity of the antibody ofthe present invention to the Aβ(20-42) globulomer is at least 2 times,e. g. at least 3 times or at least 5 times, preferably at least 10times, e. g. at least 20 times, at least 30 times or at least 50 times,more preferably at least 100 times, e. g. at least 200 times, at least300 times or at least 500 times, and even more preferably at least 1000times, e. g. at least 2000 times, at least 3000 times or at least 5000times, even more preferably at least 10000 times, e. g. at least 20000times, at least 30000 or at least 50000 times, and most preferably atleast 100000 times greater than the binding affinity of the antibody toone or, more preferably, both monomers.

Antibodies of the present invention having a comparatively smalleraffinity for at least one non-globulomer form of Aβ than for at leastone Aβ globulomer further include antibodies having a binding affinityto the Aβ(20-42) globulomer that is greater than to Aβ(1-42) fibrils.Further, it is preferred that, alternatively or additionally, thebinding affinity of the antibody to the Aβ(20-42) globulomer is greaterthan to Aβ(1-40) fibrils.

The term “fibril” here refers to a molecular structure that comprisesassemblies of noncovalently associated, individual Aβ(X-Y) peptides,which show fibrillary structure in the electron microscope, which bindCongo red and then exhibit birefringence under polarized light and whoseX-ray diffraction pattern is a cross-R structure.

In another aspect of the invention, a fibril is a molecular structureobtainable by a process that comprises the self-induced polymericaggregation of a suitable Aβ peptide in the absence of detergents, e. g.In 0.1 M HCl, leading to the formation of aggregates of more than 24,preferably more than 100 units. This process is well known in the art.Expediently, Aβ(X-Y) fibrils are used in the form of an aqueoussolution. In a particularly preferred embodiment of the invention, theaqueous fibril solution is made by dissolving the Aβ peptide in 0.1%NH₄OH, diluting it 1:4 with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4, followedby readjusting the pH to 7.4, incubating the solution at 37° C. for 20h, followed by centrifugation at 10000 g for 10 min and resuspension in20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4.

The term “Aβ(X-Y) fibril” here refers to a fibril consisting essentiallyof Aβ(X-Y) subunits, where it is preferred if on average at least 90% ofthe subunits are of the Aβ(X-Y) type, more preferred if at least 98% ofthe subunits are of the Aβ(X-Y) type, and most preferred if the contentof non-Aβ(X-Y) peptides is below the detection threshold.

More specifically, the term “Aβ(1-42) fibril” here refers to a Aβ(1-42)fibril preparation as described in example 3 herein.

Expediently, the antibody of the present invention binds to one or, morepreferably, both fibrils with low affinity, most preferably with a K_(D)of 1×10⁻⁸ M or smaller affinity, e. g. with a K_(D) of 3×10⁻⁸ M orsmaller affinity, with a K_(D) of 1×10⁻⁷ M or smaller affinity, e. g.with a K_(D) of 3×10⁻⁷ M or smaller affinity, or with a K_(D) of 1×10⁻⁶M or smaller affinity, e. g. with a K_(D) of 3×10⁻⁵ M or smalleraffinity, or with a K_(D) of 1×10⁻⁵ M or smaller affinity.

It is especially preferred that the binding affinity of the antibody ofthe present invention to Aβ(20-42) globulomer is at least 2 times, e. g.at least 3 times or at least 5 times, preferably at least 10 times, e.g. at least 20 times, at least 30 times or at least 50 times, morepreferably at least 100 times, e. g. at least 200 times, at least 300times or at least 500 times, and even more preferably at least 1000times, e. g. at least 2000 times, at least 3000 times or at least 5000times, even more preferably at least 10000 times, e. g. at least 20000times, at least 30000 or at least 50000 times, and most preferably atleast 100000 times greater than the binding affinity of the antibody toone or, more preferably, both fibrils.

According to one particular embodiment, the invention relates toantibodies having a binding affinity to the Aβ(20-42) globulomer whichis greater than its binding affinity to both Aβ(1-40) and Aβ(1-42)fibrils.

According to a particularly preferred embodiment, the present inventionrelates to antibodies having a comparatively smaller affinity for boththe monomeric and fibrillary forms of Aβ than for at least one Aβglobulomer, in particular Aβ(20-42) globulomer. These antibodieshereinafter are referred to globulomer-specific antibodies.

Antibodies of the present invention further include antibodies having abinding affinity to the Aβ(20-42) globulomer that is greater than to across-linked Aβ(1-42) globulomer, in particular to a glutardialdehydecross-linked Aβ(1-42) globulomer, such as that described in example 1bherein.

In a particularly preferred embodiment of the invention, the bindingaffinity of the antibody to Aβ(20-42) globulomer is at least 2 times, e.g. at least 3 times or at least 5 times, preferably at least 10 times,e. g. at least 20 times, at least 30 times or at least 50 times, morepreferably at least 100 times, e. g. at least 200 times, at least 300times or at least 500 times, and even more preferably at least 1000times, e. g. at least 2000 times, at least 3000 times or at least 5000times, even more preferably at least 10000 times, e. g. at least 20000times, at least 30000 or at least 50000 times, and most preferably atleast 100000 times greater than the binding affinity of the antibody tocross-linked Aβ(1-42) globulomer.

The antibodies of the present invention are preferably isolated, inparticular monoclonal and more particularly recombinant.

The present invention also relates to a monoclonal antibody (5F7) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7241.

The present invention also relates to a monoclonal antibody (10F11) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7239.

The present invention also relates to a monoclonal antibody (7C6) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7240.

The present invention also relates to a monoclonal antibody (4B7) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7242.

The present invention also relates to a monoclonal antibody (6A2) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7409.

The present invention also relates to a monoclonal antibody (2F2) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7408 The present invention also relates toa monoclonal antibody (4D10) that is obtainable from a hybridomadesignated by American Type Culture Collection deposit number PTA-7405.

The present invention also relates to a monoclonal antibody (7E5) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7809.

The present invention also relates to a monoclonal antibody (10C1) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7810.

The present invention also relates to a monoclonal antibody (3B10) thatis obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7851.

These antibodies of the present Invention, 5F7, 10F11, 7C6, 4B7, 6A2,2F2, 4D10, 7E5, 10C1 and 3B10, are characterized by having a bindingaffinity to an Aβ(20-42) globulomer that is greater than the bindingaffinity of the antibody to an Aβ(1-42) globulomer.

The present invention also relates to antibodies having a similarbinding profile to that of any one of said monoclonal antibodies, 5F7,10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10. Antibodies having asimilar binding profile to that of any one of said monoclonal antibodiesshould be understood as not being limited to antibodies having a bindingaffinity to an Aβ(20-42) globulomer that is greater than the bindingaffinity of the antibody to an Aβ(1-42) globulomer.

Antibodies having a binding profile similar to that of any one of saidmonoclonal antibodies, 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1and 3B10, include antibodies which bind to the same epitope asmonoclonal antibody 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and3B10.

All monoclonal antibodies from the group consisting of 5F7, 10F11, 7C6,4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10 bind to an epitope containedwithin the 20 and 42 Aβ sequence range, in particular within the 20-30Aβ sequence range. Without being bound to theory, said epitope isbelieved to be a structural, non-linear epitope in between subunits inthe region of amino acids 20 and 42, in particular in the region ofamino acids 20 and 30.

The present invention also relates to antibodies which are capable ofcompeting with at least one, preferably all, antibodies selected fromthe group consisting of 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1and 3B10.

The term “competing antibodies” herein refers to any number ofantibodies targeting the same molecular or stably but non-covalentlylinked supermolecular entity, preferably the same molecule, wherein atleast one is capable of specifically reducing the measurable binding ofanother, preferably by sterically hampering the other's access to itstarget epitope or by inducing and/or stabilizing a conformation in thetarget entity that reduces the target's affinity for the other antibody,more preferably by directly blocking access to the other's targetepitope by binding to an epitope in sufficiently close vicinity of theformer, overlapping with the former or identical to the former, mostpreferably overlapping or identical, in particular identical. Twoepitopes are herein said to be “overlapping” if they share part of theirchemical structures, preferably their amino acid sequences, and to be“identical”, if their chemical structures, preferably their amino acidsequences, are identical.

Thus, the present invention also relates to antibodies whose targetepitopes are overlapping with, preferably identical to, the targetepitope of at least one of the antibodies selected from the groupconsisting of 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.

Antibodies having a similar binding profile to that of any one of saidmonoclonal antibodies, 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1and 3B10, thus further include antibodies which comprise at least aportion of the antigen-binding moiety of any one of said monoclonalantibodies, 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.Preferably, said portion comprises at least one complementarydetermining region (CDR) of any one of said monoclonal antibodies, 5F7,10F11, 7C06, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.

Thus, according to a further particular embodiment, the presentinvention relates to antibodies comprising the amino acid sequence ofthe heavy chain CDR3 and/or the amino acid sequence of the right chainCDR3 of monoclonal antibody 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5,10C1 or 3B10. Specific examples of such antibodies include those whichalso comprise the amino acid sequence of the heavy chain CDR2 and/or theamino acid sequence of the light chain CDR2 of monoclonal antibody 5F7,10F11, 7C6, 487, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10, respectively. Evenmore specifically, such antibodies include those which also comprise theamino acid sequence of the heavy chain CDR1 and/or the amino acidsequence of the light chain CDR1 of monoclonal antibody 5F7, 10F11, 7C6,487, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10, respectively.

In one aspect, the present invention thus relates to antibodiescomprising a heavy chain wherein the CDR3, CDR2 and/or CDR1 domaincomprises the amino acid sequence of the heavy chain CDR3, CDR2 and/orCDR1 of monoclonal antibody 5F7, 10F11, 7C6, 487, 6A2, 2F2, 4D10, 7E5,10C1 or 3B10.

In a further aspect, the present invention thus relates to antibodiescomprising a light chain wherein the CDR3, CDR2 and/or CDR1 domaincomprises the amino acid sequence of the light chain CDR3, CDR2 and/orCDR1, respectively, of monoclonal antibody 5F7, 10F11, 7C6, 4B7, 6A2,2F2, 4D10, 7E5, 10C1 or 3B10.

Preferably, the antibody comprises at least one CDR comprising an aminoacid sequence selected from the group consisting of: amino acid residues31-35 of SEQ ID NO:3, amino acid residues 50-66 of SEQ ID NO:3, aminoacid residues 99-109 of SEQ ID NO:3, amino acid residues 24-39 of SEQ IDNO:4, amino acid residues 55-61 of SEQ ID NO:4, amino acid residues94-102 of SEQ ID NO:4, amino acid residues 31-35 of SEQ ID NO:7, aminoacid residues 50-66 of SEQ ID NO:7, amino acid residues 97-109 of SEQ IDNO:7, amino acid residues 24-39 of SEQ ID NO:8, amino acid residues55-61 of SEQ ID NO:8, amino add residues 94-102 of SEQ ID NO:8, aminoacid residues 31-35 of SEQ ID NO:11, amino acid residues 50-65 of SEQ IDNO:11, amino acid residues 98-107 of SEQ ID NO: 11, amino add residues24-39 of SEQ ID NO:12, amino acid residues 55-61 of SEQ ID NO:12, aminoacid residues 94-102 of SEQ ID NO:12, amino acid residues 31-35 of SEQID NO:15, amino acid residues 50-66 of SEQ ID NO:15, amino acid residues99-107 of SEQ ID NO:15, amino add residues 24-40 of SEQ ID NO:16, aminoacid residues 56-62 of SEQ ID NO:16, amino acid residues 95-103 of SEQID NO:16, amino acid residues 31-35 of SEQ ID NO:19, amino acid residues50-66 of SEQ ID NO:19, amino acid residues 99-109 of SEQ ID NO:19, aminoacid residues 24-39 of SEQ ID NO:20, amino acid residues 55-61 of SEQ IDNO:20, amino acid residues 94-102 of SEQ ID NO:20, amino acid residues31-35 of SEQ ID NO:23, amino acid residues 50-66 of SEQ ID NO:23, aminoacid residues 99-109 of SEQ ID NO:23, amino acid residues 24-39 of SEQID NO:24, amino acid residues 55-61 of SEQ ID NO:24, amino acid residues94-102 of SEQ ID NO:24, amino acid residues 31-35 of SEQ ID NO:27, aminoacid residues 50-65 of SEQ ID NO:27, amino acid residues 98-101 of SEQID NO:27, amino acid residues 24-39 of SEQ ID NO:28, amino acid residues55-61 of SEQ ID NO:28, amino acid residues 94-102 of SEQ ID NO:28, aminoacid residues 31-35 of SEQ ID NO:31, amino acid residues 50-66 of SEQ IDNO:31, amino acid residues 99-107 of SEQ ID NO:31, amino acid residues24-40 of SEQ ID NO:32, amino acid residues 56-62 of SEQ ID NO:32, aminoacid residues 95-103 of SEQ ID NO:32, amino acid residues 31-35 of SEQID NO:35, amino acid residues 50-66 of SEQ ID NO:35, amino acid residues99-107 of SEQ ID NO:35, amino acid residues 24-40 of SEQ ID NO:36, aminoacid residues 56-62 of SEQ ID NO:36, amino acid residues 95-103 of SEQID NO:36, amino acid residues 31-35 of SEQ ID NO:38, amino acid residues50-66 of SEQ ID NO:38, and amino acid residues 98-109 of SEQ ID NO:38.

In a preferred embodiment, the antibody comprises at least 3 CDRsselected from the group consisting of the sequences disclosed above.More preferably the 3 CDRs selected are from sets of variable domainCDRs selected from the group consisting of:

VH 5F7 CDR Set VH 5F7 CDR-H1 TFYIH: residues 31-35 of SEQ ID NO: 3VH 5F7 CDR-H2 MIGRGSGNTYYNEHFKD: residues 50-66 of SEQ ID NO: 3VH 5F7 CDR-H3 AKSARAAWFAY: residues 99-109 of SEQ ID NO: 3VL 5F7 CDR Set VL 5F7 CDR-L1 RSSQSVVQSNGNTYLE:residues 24-39 of SEQ ID NO: 4 VL 5F7 CDR-L2 KVSNRFS:residues 55-61 of SEQ ID NO: 4 VL 5F7 CDR-L3 FQGSHVPPT:residues 94-102 of SEQ ID NO: 4 VH 10F11 CDR Set VH 10F11 CDR-H1 SYVMH:residues 31-35 of SEQ ID NO: 7 VH 10F11 CDR-H2 YIYPYNDGTKYNEKFKG:residues 50-66 of SEQ ID NO: 7 VH 10F11 CDR-H3 TVEGATWDGYFDV:residues 97-109 of SEQ ID NO: 7 VL 10F11 CDR Set VL 10F11 CDR-L1KSSQSLLYSKGKTYLN: residues 24-39 of SEQ ID NO: 8 VL 10F11 CDR-L2LVSKLDS: residues 55-61 of SEQ ID NO: 8 VL 10F11 CDR-L3 VQGT5FP5T:residues 94-102 of SEQ ID NO: 8 VH 7C6 CDR Set VH 7C6 CDR-L1 SYAMS:residues 31-35 of SEQ ID NO: 11 VH 7C6 CDR-L2 SIHNRGTIFYLDSVKG:residues 50-65 of SEQ ID NO: 11 VH 7C6 CDR-L3 GRSNSYAMDY:residues 98-107 of SEQ ID NO: 11 VL 7C6 CDR Set VL 7C6 CDR-L1RSTQTLVHRNGDTYLE: residues 24-39 of SEQ ID NO: 12 VL 7C6 CDR-L2 KVSNRFS:residues 55-61 of SEQ ID NO: 12 VL 7C6 CDR-L3 FQGSHVPYT:residues 94-102 of SEQ ID NO: 12 VH 4B7 CDR Set VS 4B7 CDR-H1 DYEMV:residues 31-35 of SEQ ID NO: 15 VH 4B7 CDR-H2 YISSGSRTIHYADTVKG:residues 50-66 of SEQ ID NO: 15 VH 4B7 CDR-H3 TLLRLHFDY:residues 99-107 of SEQ ID NO: 15 VI 4B7 CDR Set VL 4B7 CDR-L1RSSQSLFYRSNQKNFLA: residues 24-40 of SEQ ID NO: 16 VL 4B7 CDR-L2WAST RES: residues 56-62 of SEQ ID NO: 16 VL 4B7 CDR-L3 QQYYSYPST:residues 95-103 of SEQ ID NO: 16 VH 2F2 CDR Set VH 2F2 CDR-H1 TFYH:residues 31-35 of SEQ ID NO: 19 VH 2F2 CDR-52 MIGPGSGNTYYNEMFKD:residues 50-66 of SEQ ID NO: 19 VH 2F2 CDR-53 AKSARAAWFAY:residues 99-109 of SEQ ID NO: 19 VL 2F2 CDR Set VL 2F2 CDR-L1PSSQSVVQSNGNTYLE: residues 24-39 of SEQ ID NO: 20 VL 2F2 CDR-L2 KVSNRFS:residues 55-61 of SEQ ID NO: 20 VL 2F2 CDR-L3 FQGSHVPPT:residues 94-102 of SEQ ID NO: 20 VH 6A2 CDR Set VH 6A2 CDR-H1 TFYIH:residues 31-35 of SEQ ID NO: 23 VH 6A2 CDR-H2 MIGPGSGNTYYNEMFKD:residues 50-66 of SEQ ID NO: 23 VH 6A2 CDR-H3 AKSHRAAWFAY:residues 99-109 of SEQ ID NO: 23 VL 6A2 CDR Set VL 6A2 CDR-L1RSSQSVVQSNGNTYLE: residues 24-39 of SEQ ID NO: 24 VL 6A2 CDR-L2 KVSNRFF:residues 55-61 of SEQ ID NO: 24 VL 6A2 CDR-L3 FQGSHVPPT:residues 94-102 of SEQ ID NO: 24 VH 4D10 CDR Set VH 4D10 CDR-H1 SYGVH:residues 31-35 of SEQ ID NO: 27 VH 4D10 CDR-H2 VIWRGGRIDYNAAFMS:residues 50-65 of SEQ ID NO: 27 VH 4D10 CDR-H3 NSDV:residues 98-101 of SEQ ID NO: 27 VL 4D10 CDR Set VL 4D10 CDR-L1KSSQSLLDIDGKTYLN: residues 24-39 of SEQ ID NO: 28 VL 4D10 CDR-L2LVSKLDS: residues 55-61 of SEQ ID NO: 28 VL 4D10 CDR-L3 WQGTHFPYT:residues 94-102 of SEQ ID NO: 28 VH 7E5 CDR Set VH 7E5 CDR-H1 DYEMV:residues 31-35 of SEQ ID NO: 31 VH 7E5 CDR-H2 YISSGSRTIHYADTVKG:residues 50-66 of SEQ ID NO: 31 VH 7E5 CDR-H3 TLLRLHFDY:residues 99-107 of SEQ ID NO: 31 VL 7E5 CDR Set VL 7E5 CDR-L1RSSQSLFYRSNQKNFLA: residues 24-40 of SEQ ID NO: 32 VL 7E5 CDR-L2WASTRES: residues 56-62 of SEQ ID NO: 32  VL 7E5 CDR-L3 QQYYSYPWT:residues 95-103 of SEQ ID NO: 32 VH 10C1 CDR Set VH 10C1 CDR-H1 DYEMV:residues 31-35 of SEQ ID NO: 35 VH 10C1 CDR-H2 YINSGSGTIHYADTVKG:residues 50-66 of SEQ ID NO: 35 VH 10C1 CDR-H3 TLLRLHFDY:residues 99-107 of SEQ ID NO: 35 VL 10C1 CDR Set VL 10C1 CDR-L1KSSQEFYSRNQKNFLA: residues 24-40 of SEQ ID NO: 36 VL 10C1 CDR-L2WASTGES: residues 56-62 of SEQ ID NO: 36 VL 10C1 CER-L3 QQYFSYWT:residues 95-103 of SEQ ID NO: 36 VH 3B10 CDR Sat VH 3B10 CDR-H1 DYVIH:residues 31-35 of SEQ ID NO: 38 VH 3B10 CDR-H2 YINPYNDGTQYNEKFKG:residues 50-66 of SEQ ID NO: 38 VH 3B10 CDR-H3 VEGGTWDGYFDV:residues 98-109 of SEQ ID NO: 38

In one embodiment the antibody of the invention comprises at least twovariable domain CDR sets. More preferably, the two variable domain CDRsets are selected from the group consisting of: VH 5F7 CDR Set & VL 5F7CDR Set; VH 10F11 CDR Set & VL 10F11 CDR Set; VH 7C6 CDR Set & VL 7C6CDR Set; VH 4B7 CDR Set & VL 4B7 CDR Set; VH 2F2 CDR Set & VL 2F2 CDRSet; VH 6A2 CDR Set & VL 6A2 CDR Set; VH 4D10 CDR Set & VL 4010 CDR Set;VH 7E5 CDR Set & VL 7E5 CDR Set; and VH 10C1 CDR Set & VL 10C1 CDR Set.

In another embodiment the antibody disclosed above further comprises ahuman acceptor framework.

In a preferred embodiment the antibody is a CDR grafted antibody.Preferably the CDR grafted antibody comprises one or more of the CDRsdisclosed above.

Preferably the CDR grafted antibody comprises a human acceptorframework.

In a preferred embodiment the antibody is a humanized antibody.Preferably the humanized antibody comprises one or more of the CDRsdisclosed above. More preferably the humanized antibody comprises threeor more of the CDRs disclosed above. Most preferably the humanizedantibody comprises six CDRs disclosed above. In a particular embodiment,the CDRs are incorporated into a human antibody variable domain of ahuman acceptor framework. Preferably the human antibody variable domainis a consensus human variable domain. More preferably the human acceptorframework comprises at least one Framework Region amino acidsubstitution at a key residue, wherein the key residue is selected fromthe group consisting of a residue adjacent to a CDR; a glycosylationsite residue; a rare residue; a residue capable of interacting withAβ(20-42) globulomer; a residue capable of interacting with a CDR; acanonical residue; a contact residue between heavy chain variable regionand light chain variable region; a residue within a Vernier zone; and aresidue in a region that overlaps between a Chothia-defined variableheavy chain CDR1 and a Kabat-defined first heavy chain framework.Preferably the human acceptor framework human acceptor frameworkcomprises at least one Framework Region amino acid substitution, whereinthe amino acid sequence of the framework is at least 65% identical tothe sequence of said human acceptor framework and comprises at least 70amino acid residues identical to said human acceptor framework.

In yet a further aspect, the present invention relates to antibodiescomprising both the heavy and light chain as defined above.

Preferably, the antibody comprises at least one variable domain havingan amino acid sequence selected from the group consisting of: SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:38. More preferably, theantibody comprises two variable domains, wherein said two variabledomains have amino acid sequences selected from the group consisting of:SEQ ID NO:3 & SEQ ID NO:4, SEQ ID NO:7 & SEQ ID NO:8 & SEQ ID NO:11 &SEQ ID NO:12, SEQ ID NO:15 & SEQ ID NO:16, SEQ ID NO:19 & SEQ ID NO:20,SEQ ID NO:23 & SEQ ID NO:24, SEQ ID NO:27 & SEQ ID NO:28, SEQ ID NO:31 &SEQ ID NO:32, and SEQ ID NO:35 & SEQ ID NO:36.

In another aspect, the antibodies of the present invention comprise aheavy chain constant region selected from the group consisting of IgG1,IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and human IgG1 Ala234 Ala235 mutantconstant regions. In particular, the antibodies comprise a humanconstant region. More preferably, the antibodies comprise an amino addsequence selected from the group consisting of SEQ ID NOs:39-42.Antibodies comprising an IgG 1 heavy chain constant region arepreferred.

In another embodiment the antibody is glycosylated. Preferably theglycosylation pattern is a human glycosylation pattern or aglycosylation pattern produced by any one of the eukaryotic cellsdisclosed herein, in particular CHO cells.

The present invention also relates to an antigen-binding moiety of anantibody of the present invention. Such antigen-binding moietiesinclude, but are not limited to, Fab fragments, F(ab′)₂ fragments andsingle chain Fv fragments of the antibody. Further antigen-bindingmoieties are Fab′ fragments, Fv fragments, and disulfide linked Fvfragments.

The invention also provides an isolated nucleic acid encoding any one ofthe antibodies disclosed herein. A further embodiment provides a vectorcomprising the isolated nucleic acid disclosed herein. Said vector mayin particular be selected from the group consisting of pcDNA; pTT(Durocher et al., Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTTwith additional multiple cloning site; pEFBOS (Mizushima, S. and Nagata,S., (1990) Nucleic Acids Research Vol 18, No. 17); pBV; pJV; and pBJ.

In another aspect a host cell is transformed with the vector disclosedherein. Preferably the host cell is a prokaryotic cell. More preferablythe host cell is E coll. In a related embodiment the host cell is aeukaryotic cell. Preferably the eukaryotic cell is selected from thegroup consisting of a protist cell, an animal cell (e g. a mammaliancell, an avian cell, and an insect cell), a plant cell and a fungalcell. More preferably the host cell is a mammalian cell including, butnot limited to, CHO and COS; or a fungal cell, eg., a yeast cell, suchas Saccharomyces cerevisiae; or an insect cell such as Sf9.

Another aspect of the invention provides a method of producing anantibody of the invention, comprising culturing any one of the hostcells or a hybridoma disclosed herein in a culture medium underconditions suitable to produce the antibody. Another embodiment providesan antibody that is obtainable by the method disclosed herein.

Antibodies of the present invention can be obtained in a manner knownper se.

B lymphocytes which, in totality, contain an antibody repertoirecomposed of hundreds of billions of different antibody specificities area part of the mammalian immune system. A normal immune response to aparticular antigen means selection of one or more antibodies of saidrepertoire which specifically bind to said antigen, and the success ofan immune response is based at least partially on the ability of saidantibodies to specifically recognize (and ultimately to eliminate) thestimulating antigen and to ignore other molecules in the environment ofsaid antibodies.

The usefulness of antibodies which specifically recognize one particulartarget antigen has led to the development of monoclonal antibodytechnology. Standardized hybridoma technology now allows the productionof antibodies with a single specificity for an antigen of interest Morerecently, recombinant antibody techniques such as in-vitro screening ofantibody libraries have been developed. These techniques likewise allowantibodies having a single specificity for an antigen of interest to beproduced.

In the method of the invention, the antigen of interest may be allowedto act on the antibody repertoire either in vivo or in vitro.

According to one embodiment, the antigen is allowed to act on therepertoire by immunizing an animal in vivo with said antigen. Thisin-vivo approach may furthermore comprise establishing from thelymphocytes of an animal a number of hybridomas and selecting aparticular hybridoma which secretes an antibody specifically binding tosaid antigen. The animal to be immunized may be, for example, a mouse,rat, rabbit, chicken, camelid or sheep or may be a transgenic version ofany of the animals mentioned above, for example a transgenic mouse withhuman immunoglobulin genes, which produces human antibodies after anantigenic stimulus. Other types of animals which may be immunizedinclude mice with severe combined immunodeficiency (SCID) which havebeen reconstituted with human peripheral mononuclear blood cells(chimeric hu-PBMC SCID mice) or with lymphoid cells or precursorsthereof, as well as mice which have been treated with a lethal totalbody irradiation, then protected against radiation with bone marrowcells from a mouse with severe combined immunodeficiency (SCID) andsubsequently transplanted with functional human lymphocytes (the“Trimera” system). Another type of an animal to be immunized is ananimal (e.g. a mouse) in whose genome an endogenous gene encoding theantigen of interest has been switched off (knocked out), for example byhomologous recombination, so that, after immunization with the antigen,said animal recognizes said antigen as foreign. It is obvious to theskilled worker that the polyclonal or monoclonal antibodies produced bythis method are characterized and selected by using known screeningmethods which include, but are not limited to, ELISA and dot blottechniques.

According to another embodiment, the antigen is allowed to act on theantibody repertoire in vitro by screening a recombinant antibody librarywith said antigen. The recombinant antibody library may be expressed,for example, on the surface of bacteriophages or on the surface of yeastcells or on the surface of bacterial cells. In a variety of embodiments,the recombinant antibody library is an scFv library or an Fab library,for example. According to another embodiment, antibody libraries areexpressed as RNA-protein fusions.

Another approach to producing antibodies of the Invention comprises acombination of in vivo and in vitro approaches. For example, the antigenmay be allowed to act on the antibody repertoire by immunizing an animalin vivo with said antigen and then screening in vitro with said antigena recombinant antibody library prepared from lymphoid cells of saidanimal or a single domain antibody library (e.g. containing heavy and/orlight chains). According to another approach, the antigen is allowed toact on the antibody repertoire by immunizing an animal in vivo with saidantigen and then subjecting a recombinant antibody library or singledomain library produced from lymphoid cells of said animal to affinitymaturation. According to another approach, the antigen is allowed to acton the antibody repertoire by immunizing an animal in vivo with saidantigen, then selecting individual antibody-producing cells secreting anantibody of interest and obtaining from said selected cells cDNAs forthe variable region of the heavy and light chains (e.g. by means of PCR)and expressing said variable regions of the heavy and light chains inmammalian host cells in vitro (this being referred to as selectedlymphocyte antibody method or SLAM), thereby being able to furtherselect and manipulate the selected antibody gene sequences. Moreover,monoclonal antibodies may be selected by expression cloning byexpressing the antibody genes for the heavy and light chains inmammalian cells and selecting those mammalian cells which secrete anantibody having the desired binding affinity.

The present invention provides defined antigens for screening andcounter screening. Thus it is possible, according to the invention, toselect those polyclonal and monoclonal antibodies which bind to anAβ(20-42) globulomer with the binding affinities as defined above.

The methods of the invention for producing antibodies can be used toproduce various types of antibodies. These include monoclonal, inparticular recombinant antibodies, especially essentially humanantibodies, chimeric antibodies, humanized antibodies and CDR graftantibodies, and also antigen-binding moieties thereof.

The present invention further relates to a hybridoma that is capable ofproducing (secreting) a monoclonal antibody of the present invention.Hybridomas of the present invention include those designated by anAmerican Type Culture Collection deposit number selected from the groupconsisting of PTA-7241, PTA-7239, PTA-7240, PTA-7242, PTA-7408,PTA-7409, PTA-7405, PTA-7809, PTA-7810 and PTA-7851.

It is noted that the antibodies of the present invention may also bereactive with, i.e. bind to, AP forms other than the Aβ globulomersdescribed herein. These antigens may or may not be oligomeric orglobulomeric. Thus, the antigens to which the antibodies of the presentinvention bind include any Aβ form that comprises the globulomer epitopewith which the antibodies of the present invention are reactive. Such Aβforms include truncated and non-truncated Aβ(X-Y) forms (with X and Ybeing defined as above), such as Aβ(20-42), Aβ(20-40), Aβ(12-42),Aβ(12-40), Aβ(1-42), and Aβ(1-40) forms, provided that said formscomprise the globulomer epitope.

The present invention also relates to a composition comprising anantibody of the invention or an antigen-binding moiety thereof, asdefined above.

According to a particular embodiment, said composition is apharmaceutical composition which comprises the antibody of the Inventionor the antigen-binding moiety and a pharmaceutical acceptable carrier.

The antibody of the invention or the antigen-binding moiety as definedabove is preferably capable of neutralizing, both in vitro and in vivo,the activity of Aβ globulomer or a derivative thereof to which it binds.Said antibody or antigen-binding moiety may therefore be used forInhibiting the activity of said globulomer or derivative thereof, forexample in a preparation containing said globulomer or derivativethereof or in human individuals or other mammals in which saidglobulomer or derivative thereof is present.

According to one embodiment, the invention relates to a method ofinhibiting the activity of said globulomer or derivative thereof, whichmethod comprises allowing an antibody of the invention or anantigen-binding moiety thereof to act on a globulomer or derivativethereof so as to inhibit the activity of said globulomer or derivativethereof. Said activity may be inhibited in vitro, for example. Forinstance, the antibody of the invention or the antigen-binding moietymay be added to a preparation such as a sample derived from a subject ora cell culture which contains or is suspected to contain said globulomeror derivative thereof, in order to inhibit the activity of saidglobulomer or derivative thereof in said sample. Alternatively, theactivity of the globulomer or derivative thereof may be Inhibited in anindividual in vivo.

Thus the present invention further relates to the use of an antibody oran antigen-binding moiety as defined above for preparing apharmaceutical composition for treating or preventing an amyloidosis, inparticular an amyloidosis selected from the group consisting ofAlzheimer's disease and the amyloidosis of Down's syndrome. One aspectof said use of the invention is therefore a method of treating orpreventing an amyloidosis, in particular Alzheimer's disease or theamyloidosis of Down's syndrome, in a subject in need thereof, whichcomprises administering an antibody or an antigen-binding moiety asdefined above to the subject. Using said antibody or antigen-bindingmoiety for treating and especially preventing the amyloidosis, inparticular Alzheimer's disease or the amyloidosis of Down's syndrome, isin particular for passive immunization. Accordingly, in the method oftreating or preventing an amyloidosis, in particular Alzheimer's diseaseor the amyloidosis of Down's syndrome, in a subject in need thereof onepurpose of administering the antibody or antigen-binding moiety to thesubject is passively immunizing the subject against the amyloidosis, inparticular Alzheimer's disease or the amyloidosis of Down's syndrome.

The antibody of the Invention or the antigen-binding moiety as definedabove is preferably capable of detecting, both in vitro and in vivo, anAβ globulomer or derivative thereof to which it binds. Said antibody orthe antigen-binding moiety may therefore be used for detecting saidglobulomer or derivative thereof, for example in a preparationcontaining said globulomer or derivative thereof or in human individualsor other mammals in which said globulomer or derivatives thereof ispresent.

According to one embodiment, the invention relates to a method ofdetecting said globulomer or derivative thereof, which method comprisesallowing an antibody of the invention or an antigen-binding moietythereof to act on a globulomer or derivative thereof so as to bind tosaid globulomer or derivative thereof (and thereby preferably forming acomplex comprising the antibody or antigen-binding moiety thereof andthe globulomer or derivative thereof). Said globulomer may be detectedin vitro, for example. For example, the antibody of the invention or theantigen-binding moiety may be added to a preparation, for instance asample derived from a subject or a cell culture which contains or issuspected to contain said globulomer or derivative thereof, in order todetect said globulomer or derivative thereof in said preparation.Alternatively, the globulomer or derivative thereof may be detected inan individual in vivo.

Thus the present invention further relates to the use of an antibody oran antigen-binding moiety as defined above for preparing a compositionfor diagnosing an amyloidosis, in particular Alzheimer's disease or theamyloidosis of Down's syndrome. One aspect of said use of the inventionis a method of diagnosing an amyloidosis, in particular Alzheimer'sdisease or the amyloidosis of Down's syndrome, in a subject suspect ofhaving the amyloidosis, in particular Alzheimer's disease or theamyloidosis of Down's syndrome, which comprises administering to thesubject an antibody or an antigen-binding moiety as defined above anddetecting the formation of a complex comprising the antibody or theantigen-binding moiety with the antigen, the presence of the complexIndicating the amyloidosis, in particular Alzheimer's disease or theamyloidosis of Down's syndrome, In the subject. A second aspect of saiduse of the invention is a method of diagnosing an amyloidosis, inparticular Alzheimer's disease or the amyloidosis of Down's syndrome, ina subject suspect of having the amyloidosis, in particular Alzheimer'sdisease or the amyloidosis of Down's syndrome, which comprises providinga sample from the subject, contacting the sample with an antibody or anantigen-binding moiety as defined above and detecting the formation of acomplex comprising the antibody or the antigen-binding moiety with theantigen, the presence of the complex indicating the amyloidosis, inparticular Alzheimer's disease or the amyloidosis of Down's syndrome, inthe subject

DETAILED DESCRIPTION OF THE INVENTION

The binding affinities of the antibodies of the invention may beevaluated by using standardized in-vitro immunoassays such as ELISA, dotblot or BIAcore analyses (Pharmacia Biosensor AB, Uppsala, Sweden andPiscataway, N.J.). For further descriptions, see Jönsson, U., et al.(1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991)BioTechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

According to a particular embodiment, the affinities defined hereinrefer to the values obtained by performing a dot blot as described inexample 8 and evaluating it by densitometry. According to a particularembodiment of the invention, determining the binding affinity by dotblot comprises the following: a certain amount of the antigen (e.g. theAβ(X-Y) globulomer, Aβ(X-Y) monomer or Aβ(X-Y) fibrils, as definedabove) or, expediently, an appropriate dilution thereof, for instance in20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA to an antigenconcentration of, for example, 100 pmol/μl, 10 pmol/μl, 1 pmol/μl, 0.1pmol/μl and 0.01 pmol/μl, is dotted onto a nitrocellulose membrane, themembrane is then blocked with milk to prevent unspecific binding andwashed, then contacted with the antibody of interest followed bydetection of the latter by means of an enzyme-conjugated secondaryantibody and a colorimetric reaction; at defined antibodyconcentrations, the amount of antibody bound allows affinitydetermination. Thus the relative affinity of two different antibodies toone target, or of one antibody to two different targets, is here definedas the relation of the respective amounts of target-bound antibodyobserved with the two antibody-target combinations under otherwiseidentical dot blot conditions. Unlike a similar approach based onWestern blotting, the dot blot approach will determine an antibody'saffinity to a given target in the latter's natural conformation; unlikethe ELISA approach, the dot blot approach does not suffer fromdifferences in the affinities between different targets and the matrix,thereby allowing for more precise comparisons between different targets.

The term “K_(d)”, as used herein, is Intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The antibodies of the present invention are preferably isolatedantibodies. An “isolated antibody” means an antibody having the bindingaffinities as described above and which is essentially free of otherantibodies having different binding affinities. The term “essentiallyfree” here refers to an antibody preparation in which at least 95% ofthe antibodies, preferably at least 98% of the antibodies and morepreferably at least 99% of the antibodies have the desired bindingaffinity. Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The isolated antibodies of the present invention include monoclonalantibodies. A “monoclonal antibody” as used herein is intended to referto a preparation of antibody molecules, antibodies which share a commonheavy chain and common light chain amino acid sequence, in contrast with“polyclonal” antibody preparations which contain a mixture of antibodiesof different amino acid sequence. Monoclonal antibodies can be generatedby several novel technologies like phage, bacteria, yeast or ribosomaldisplay, as well as by classical methods exemplified byhybridoma-derived antibodies (e.g., an antibody secreted by a hybridomaprepared by hybridoma technology, such as the standard Kohler andMilstein hybridoma methodology ((1975) Nature 256:495-497). Thus, anon-hybridoma-derived antibody with uniform sequence is still referredto as a monoclonal antibody herein although it may have been obtained bynonclassical methodologies, and the term “monoclonal” is not restrictedto hybridoma-derived antibodies but used to refer to all antibodiesderived from one nucleic acid clone.

Thus, the monoclonal antibodies of the present invention includerecombinant antibodies. The term “recombinant” herein refers to anyartificial combination of two otherwise separated segments of sequence,e.g., by chemical synthesis or by the manipulation of isolated segmentsof nucleic acids by genetic engineering techniques. In particular, theterm “recombinant antibody” refers to antibodies which are produced,expressed, generated or isolated by recombinant means, such asantibodies which are expressed using a recombinant expression vectortransfected into a host cell; antibodies isolated from a recombinantcombinatorial antibody library; antibodies isolated from an animal (e.g.a mouse) which is transgenic due to human immunoglobulin genes (see, forexample, Taylor, L. D., et al. (1992) Nucl Acids Res 20:6287-6295); orantibodies which are produced, expressed, generated or isolated in anyother way in which particular immunoglobulin gene sequences (such ashuman immunoglobulin gene sequences) are assembled with other DNAsequences. Recombinant antibodies include, for example, chimeric, CDRgraft and humanized antibodies. The person skilled in the art will beaware that expression of a conventional hybridoma-derived monoclonalantibody in a heterologous system will require the generation of arecombinant antibody even if the amino acid sequence of the resultingantibody protein is not changed or Intended to be changed.

In a particular embodiment of the invention, the antibody is a humanizedantibody.

According to a multiplicity of embodiments, the antibody may comprise anamino acid sequence derived entirely from a single species, such as ahuman antibody or a mouse antibody. According to other embodiments, theantibody may be a chimeric antibody or a CDR graft antibody or anotherform of a humanized antibody.

The term “antibody” is intended to refer to immunoglobulin moleculesconsisting of 4 polypeptide chains, two heavy (H) chains and two light(L) chains. The chains are usually linked to one another via disulfidebonds. Each heavy chain is composed of a variable region of said heavychain (abbreviated here as HCVR or VH) and a constant region of saidheavy chain. The heavy chain constant region consists of three domainsCH1, CH2 and CH3. Each light chain is composed of a variable region ofsaid light chain (abbreviated here as LCVR or VL) and a constant regionof said light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region thus consists of three CDRs and four FRswhich are arranged from the N terminus to the C terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell known to those skilled in the art.

The term “antigen-binding moiety” of an antibody (or simply “antibodymoiety”) refers to one or more fragments of an antibody of theinvention, said fragment(s) still having the binding affinities asdefined above. Fragments of a complete antibody have been shown to beable to carry out the antigen-binding function of an antibody. Inaccordance with the term “antigen-binding moiety” of an antibody,examples of binding fragments include (i) an Fab fragment, i.e. amonovalent fragment composed of the VL, VH, CL and CH1 domains; (ii) anF(ab′)₂ fragment, i.e. a bivalent fragment comprising two Feb fragmentslinked to one another in the hinge region via a disulfide bridge; (iii)an Fd fragment composed of the VH and CH1 domains; (iv) an Fv fragmentcomposed of the FL and VH domains of a single arm of an antibody; (v) adAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VHdomain or of VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) an isolatedcomplementarity-determining region (CDR). Although the two domains ofthe Fv fragment, namely VL and VH, are encoded by separate genes, theymay further be linked to one another using a synthetic linker, e.g. apoly-G₄S amino acid sequence, and recombinant methods, making itpossible to prepare them as a single protein chain in which the VL andVH regions combine in order to form monovalent molecules (known assingle chain Fv (ScFv); see, for example, Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Nat. Acad. Sci. USA85:5879-5883). The term “antigen-binding moiety” of an antibody is alsointended to comprise such single chain antibodies. Other forms of singlechain antibodies such as “diabodies” are likewise included here.Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker which istoo short for the two domains being able to combine on the same chain,thereby forcing said domains to pair with complementary domains of adifferent chain and to form two antigen-binding sites (see, for example,Holliger, P., et al. (1993) Proc. Natl. Acad. Sc. USA 90:6444-6448;Poljak, R. J., et a. (1994) Structure 2:1121-1123). An immunoglobulinconstant domain refers to a heavy or light chain constant domain. HumanIgG heavy chain and light chain constant domain amino acid sequences areknown in the art and are represented in Table 1.

TABLE 1 Sequence of human IgG heavy chain constant domain and light chainconstant domain Sequence Protein Sequence ID12345678901234567890123456799012 Ig gamma-1 SEQ ID NO: 39ASTKGPSVFFLAPSSKSTSGGTAALGCLVKDY constant regionFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 SEQ ID NO: 40ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant regionFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutant LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig Kappa constantSEQ ID NO: 41 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY regionPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda SEQ ID NO: 42 QPKAAPSVTLFPPSSEELQANKATLVCLISDFconstant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS

Furthermore, an antibody of the present invention or antigen-bindingmoiety thereof may be part of a larger immunoadhesion molecule formed bycovalent or noncovalent association of said antibody or antibody moietywith one or more further proteins or peptides. Relevant to suchimmunoadhesion molecules are the use of the streptavidin core region inorder to prepare a tetrameric scFv molecule (Kipriyanov, S. M., et at(1995) Human Antibodies and Hybridomas 6:93-101) and the use of acystein residue, a marker peptide and a C-terminal polyhistidinyl, e. g.hexahistidinyl, tag in order to produce bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

The term “human antibody” refers to antibodies whose variable andconstant regions correspond to or are derived from immunoglobulinsequences of the human germ line, as described, for example, by Kabat etal. (see Kabat, et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). However, the human antibodies of theinvention may contain amino acid residues not encoded by human germ lineimmunoglobulin sequences (for example mutations which have beenintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs, and in particular in CDR3.Recombinant human antibodies of the invention have variable regions andmay also contain constant regions derived from immunoglobulin sequencesof the human germ line (see Kabat, E A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). According toparticular embodiments, however, such recombinant human antibodies aresubjected to in-vitro mutagenesis (or to a somatic in-vivo mutagenesis,if an animal is used which is transgenic due to human Ig sequences) sothat the amino acid sequences of the VH and VI regions of therecombinant antibodies are sequences which although related to orderived from VH and VL sequences of the human germ line, do notnaturally exist in vivo within the human antibody germ line repertoire.According to particular embodiments, recombinant antibodies of this kindare the result of selective mutagenesis or back mutation or of both.Preferably, mutagenesis leads to an affinity to the target which isgreater, and/or an affinity to non-target structures which is smallerthan that of the parent antibody.

The term “chimeric antibody” refers to antibodies which containsequences for the variable region of the heavy and light chains from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which containsequences of the variable region of heavy and light chains from anonhuman species (e.g. mouse, rat, rabbit, chicken, camelid, sheep orgoat) but in which at least one part of the VH and/or VL sequence hasbeen altered in order to be more “human-like”, i.e. to be more similarto variable sequences of the human germ line. One type of a humanizedantibody is a CDR graft antibody in which human CDR sequences have beeninserted into nonhuman VH and VL sequences to replace the correspondingnonhuman CDR sequences.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues not occurring at one or more specific positions of a humanantibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences There are three CDRs in eachof the variable regions of the heavy chain and of the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,in spite of great diversity at the level of amino acid sequence. Thesesub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the“L” and the “H” designates the light chain and the heavy chains regions,respectively. These regions may be referred to as Chothia CDRs, whichhave boundaries that overlap with Kabat CDRs. Other boundaries definingCDRs overlapping with the Kabat CDRs have been described by Padlan(FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly followone of the above systems, but will nonetheless overlap with the KabatCDRs, although they may be shortened or lengthened in light ofprediction or experimental findings that particular residues or groupsof residues or even entire CDRs do not significantly impact antigenbinding. The methods used herein may utilize CDRs defined according toany of these systems, although preferred embodiments use Kabat orChothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a spades different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined using differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)).One of the advantages provided by various embodiments of the presentinvention stems from the finding that germline antibody genes are morelikely than mature antibody genes are to conserve essential amino acidsequence structures characteristic of individuals in the species, henceless likely to be recognized as non-self when used in that species.

As used herein, the term “key” residues refers to certain residueswithin the variable region that have more impact on the bindingspecificity and/or affinity of an antibody, in particular a humanizedantibody. A key residue Includes, but is not limited to, one or more ofthe following: a residue that is adjacent to a CDR, a potentialglycosylation site (which can be either N- or O-glycosylation site), arare residue, a residue capable of interacting with the antigen, aresidue capable of interacting with a CDR, a canonical residue, acontact residue between heavy chain variable region and light chainvariable region, a residue within the Vemier zone, and a residue in theregion that overlaps between the Chothia definition of a variable heavychain CDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” specifically refers to anantibody or a variant, derivative, analog or fragment thereof whichimmunospecifically binds to an antigen of interest and which comprises aframework (FR) region having substantially the amino acid sequence of ahuman antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 80%, preferably at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identical to theamino acid sequence of a non-human antibody CDR A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence.Preferably, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

The humanized antibody can be selected from any class ofImmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any subclass,including without limitation IgG1, IgG2, IgG3 and IgG4.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond exactly toeither the donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus Immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see e.g., Winnaker, From Genes toClones (Veriagsgesellschaft, Weinheim, Germany 1987). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.Where two amino acids occur equally frequently, either can be includedin the consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules.

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or anycombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide” is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is connected in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptionInitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence Is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, also to the progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

Methods of producing antibodies of the invention are described below. Adistinction is made here between in-vivo approaches, in-vitro approachesor a combination of both.

Some methods of producing antibodies of the invention are describedbelow. A distinction is made here between in-vivo approaches, in-vitroapproaches or a combination of both.

In-Vivo Approaches

Depending on the type of the desired antibody, various host animals maybe used for in-vivo immunization. A host expressing Itself an endogenousversion of the antigen of interest may be used. Alternatively, it ispossible to use a host which has been made deficient in an endogenousversion of the antigen of interest. For example, mice which had beenmade deficient in a particular endogenous protein via homologousrecombination at the corresponding endogenous gene (i.e. knockout mice)have been shown to generate a humoral response to the protein with whichthey have been immunized and therefore to be able to be used forproduction of high-affinity monoclonal antibodies to the protein (see,for example, Roes, J. et al. (1995) J. Immunol. Methods 183:231-237;Lunn, M. P. et al. (2000) J. Neurochem. 75:404-412).

A multiplicity of nonhuman mammals are suitable hosts for antibodyproduction in order to produce nonhuman antibodies of the invention.They include mice, rats, chickens, camelids, rabbits, sheep and goats(and knockout versions thereof), although preference is given to micefor the production of hybridomas. Furthermore, a nonhuman host animalexpressing a human antibody repertoire may be used for producingessentially human antibodies to a human antigen with dual specificity.Nonhuman animals of this kind include transgenic animals (e.g. mice)bearing human immunoglobulin transgenes (chimeric hu-PBMC SCID mice) andhuman/mouse irradiation chimeras which are described in more detailbelow.

According to one embodiment, the animal immunized with Aβ(20-42)globulomer or derivative thereof is a nonhuman mammal, preferably amouse, which is transgenic due to human immunoglobulin genes so thatsaid nonhuman mammal makes human antibodies upon antigenic stimulation.Typically, immunoglobulin transgenes for heavy and light chains withhuman germ line configuration are introduced into such animals whichhave been altered such that their endogenous heavy and light chain lociare Inactive. If such animals are stimulated with antigen (e.g. with ahuman antigen), antibodies derived from the human immunoglobulinsequences (human antibodies) are produced. It Is possible to make fromthe lymphocytes of such animals human monoclonal antibodies by means ofstandardized hybridoma technology. For a further description oftransgenic mice with human immunoglobulins and their use in theproduction of human antibodies, see, for example, U.S. Pat. No.5,939,598, WO 96/33735, WO 96/34096, WO 98/24893 and WO 99/53049(Abgenix Inc.), and U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,569,825,U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat. No.5,661,016, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,814,318, U.S. Pat.No. 5,877,397 and WO 99/45962 (Genpharm Inc.); see also MacQuitty, J. J.and Kay, R. M. (1992) Science 257:1188; Taylor, L. D. et al. (1992)Nucleic Acids Res. 20:6287-6295; Lonberg, N. et al. (1994) Nature368:856-859; Lonberg, N. and Huszar, D. (1995) Int. Rev. Immunol.13:65-93; Harding, F. A. and Lonberg, N. (1995) Ann. N.Y. Aced. Sc.764:536-546; Fishwild, D. M. et al. (1996) Nature Biotechnology14:845-851; Mendez, M. J. et al. (1997) Nature Genetics 15:146-156;Green, L. L. and Jakobovits, A. (1998) J. Exp. Med. 188:483-495; Green,L L. (1999) J. Immunol. Methods 231:11-23; Yang, X. D. et al. (1999) J.Leukoc. Biol. 66:401-410; Gallo, M. L. et al. (2000) Eur. J. Immunol.30:534-540.

According to another embodiment, the animal which is immunized withAβ(20-42) globulomer or derivative thereof may be a mouse with severecombined immunodeficiency (SCID), which has been reconstituted withhuman peripheral mononuclear blood cells or lymphoid cells or precursorsthereof. Such mice which are referred to as chimeric hu-PBMC SCID miceproduce human immunoglobulin responses upon antigenic stimulation, ashas been proved. For a further description of these mice and of theiruse for generating antibodies, see, for example, Leader, K. A. et al.(1992) Immunology 76:229-234; Bombil, F. et al. (1996) Immunobiol.195:360-375; Murphy, W. J. et al. (1996) Semin. Immunol. 8:233-241;Herz, U. et al. (1997) Int. Arch. Allergy Immunol. 113:150-152; Albert,S. E. et al. (1997) J. Immunol. 159:1393-1403; Nguyen, H. et al. (1997)Microbiol Immunol. 41:901-907; Arai, K. et al. (1998) J. Immunol.Methods 217:79-85; Yoshinarl, K. and Arai, K. (1998) Hybridoma 17:41-45;Hutchins, W. A. et al. (1999) Hybridoma 18:121-129; Murphy, W. J. et al.(1999) Clin. Immunol 90:22-27; Smithson, S. L. et al. (1999) Mol.Immunol. 36:113-124; Chamat, S et al. (1999) J. Infect. Diseases180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:35-45.

According to another embodiment, the animal which is immunized withAβ(20-42) globulomer or a derivative thereof is a mouse which has beentreated with a lethal dose of total body irradiation, then protectedfrom radiation with bone marrow cells from mice with severe combinedimmunodeficiency (SCID) and subsequently transplanted with functionalhuman lymphocytes. This type of chimera, referred to as the Trimerasystem, is used in order to produce human monoclonal antibodies byimmunizing said mice with the antigen of interest and then producingmonoclonal antibodies by using standardized hybridoma technology. For afurther description of these mice and of their use for generatingantibodies, see, for example, Eren, R et al. (1998) Immunology93:154-161; Reisner, Y and Dagan, S. (1998) Trends Biotechnol.16:242-246; Ilan, E. et al. (1999) Hepatology 29:553-562; and Bocher, W.O. et al. (1999) Immunology 96:634-641.

Starting from the in-vivo generated antibody-producing cells, monoclonalantibodies may be produced by means of standardized techniques such asthe hybridoma technique originally described by Kohler and Milstein(1975, Nature 256:495-497) (see also Brown et al. (1981) J. Immunol127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et a. (1982) Int. J. Cancer 29:269-75).The technology of producing monoclonal antibody hybridomas issufficiently known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lemer (1981) Yale J. Biol. Med.,54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36).Briefly, an immortalized cell line (typically a myeloma) is fused withlymphocytes (typically splenocytes or lymph node cells or peripheralblood lymphocytes) of a mammal immunized with the Aβ globulomer of theinvention or derivative thereof, and the culture supernatants of theresulting hybridoma cells are screened in order to identify a hybridomawhich produces a monoclonal antibody of the present invention. Any ofthe many well known protocols for fusing lymphocytes and immortalizedcell lines can be applied for this purpose (see also G. Galfre et al.(1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., citedsupra; Lemer, Yale J. Biol. Med., cited supra; Kenneth, MonoclonalAntibodies, cited supra). Moreover, the skilled worker will appreciatethat there are diverse variations of such methods, which are likewiseuseful. Typically, the Immortalized cell line (e.g. a myeloma cell line)is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas may be established by fusing lymphocytes froma mouse immunized with an immunogenic preparation of the invention withan immortalized mouse cell line. Preferred immortalized cell lines aremouse myeloma cell lines which are sensitive to culture mediumcontaining hypoxanthine, aminopterine and thymidine (HAT medium). Any ofa number of myeloma cell lines may be used by default as fusion partner,for example the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myelomalines. These myeloma cell lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(PEG). Hybridoma cells resulting from the fusion are then selected usingHAT medium, thereby killing unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing monoclonal antibodies of theinvention are identified by screening the hybridoma culture supernatantsfor such antibodies, for example by using a dot blot assay as describedabove and in example 8 in order to select those antibodies which havethe binding affinities as defined above.

The monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5,10C1, and 3B10 all have been generated using the above-described in-vivoapproach and thereof are obtainable from a hybridoma as defined herein.

Likewise, said hybridoma can be used as a source of nucleic acidencoding light and/or heavy chains in order to recombinantly produceantibodies of the present Invention, as is described below in furtherdetail.

In-Vitro Approaches

As an alternative to producing antibodies of the invention byimmunization and selection, antibodies of the invention may beidentified and isolated by screening recombinant combinatorialimmunoglobulin libraries with Aβ(20-42) globulomer or derivative thereofto thereby isolate immunoglobulin library members which have therequired binding affinity. Kits for generating and screening displaylibraries are commercially available (e.g. the Pharmacia RecombinantPhage Antibody System, catalog No. 27-9400-01; and the StratageneSurfZAP® Phage Display Kit, catalog No. 240612). In many embodiments,the display library is an scFv library or an Fab library. The phagedisplay technique for screening recombinant antibody libraries has beenadequately described. Examples of methods and compounds which can beused particularly advantageously for generating and screening antibodydisplay libraries can be found, for example, in McCafferty et al. WO92/01047, U.S. Pat. No. 5,969,108 and EP 589 877 (describes inparticular scFv display), Ladner et al. U.S. Pat. No. 5,223,409, U.S.Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500 andEP 436 597 (describes pill fusion, for example); Dower et al. WO91/17271, U.S. Pat. No. 5,427,908, U.S. Pat. No. 5,580,717 and EP 527839 (describes in particular Fab display); Winter et al. InternationalPublication WO 92/20791 and EP 368,684 (describes in particular thecloning of sequences for variable immunoglobulin domains); Griffiths etal. U.S. Pat. No. 5,885,793 and EP 589 877 (describes in particularisolation of human antibodies to human antigens by using recombinantlibraries); Garrard et al. WO 92/09690 (describes in particular phageexpression techniques); Knappik et al. WO 97/08320 (describes the humanrecombinant antibody library HuCal); Salfeld et al. WO 97/29131,(describes production of a recombinant human antibody to a human antigen(human tumor necrosis factor alpha) and also in-vitro affinitymaturation of the recombinant antibody) and Salfeld et al U.S.Provisional Application No. 60/126,603 and the patent applications basedhereupon (likewise describes production of recombinant human antibodiesto human antigen (human interleukin-12), and also in-vitro affinitymaturation of the recombinant antibody).

Further descriptions of screenings of recombinant antibody libraries canbe found in scientific publications such as Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Bo 226:889-896;Clarkson et ael. (1991) Nature 352:624-628; Gram et al. (1992) PNAS89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377;Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991)PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; andKnappik at al. (2000) J. Mol. Biol. 296:57-86.

As an alternative to using bacteriophage display systems, recombinantantibody libraries may be expressed on the surface of yeast cells or ofbacterial cells. WO 99/36569 describes methods of preparing andscreening libraries expressed on the surface of yeast cells. WO 98/49286describes in more detail methods of preparing and screening librariesexpressed on the surface of bacterial cells.

In all in vitro approaches, a selection process for enrichingrecombinant antibodies with the desired properties form an integral partof the process, which is generally referred to as “panning” and oftentakes the form of affinity chromatography over columns to whose matrixthe target structure has been attached. Promising candidate moleculesare then subjected to individual determination of their absolute and/orrelative affinities, preferably by means of a standardized dot blotassay, as described above and in example 8.

Once an antibody of Interest of a combinatorial library has beenidentified and sufficiently characterized, the DNA sequences encodingthe light and heavy chains of said antibody are isolated by means ofstandardized molecular-biological techniques, for example by means ofPCR amplification of DNA from the display package (e.g. the phage) whichhas been isolated during library screening. Nucleotide sequences ofgenes for light and heavy antibody chains, which may be used forpreparing PCR primers, are known to the skilled worker. A multiplicityof such sequences are described, for example, in Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242 and in the database of sequences of the human germ line VBASE.

An antibody or antibody moiety of the invention may be produced byrecombinantly expressing the genes for light and heavy immunoglobulinchains in a host cell. In order to recombinantly express an antibody, ahost cell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the light and heavy immunoglobulinchains of said antibody, thereby expressing the light and heavy chainsin the host cell and secreting them preferably into the medium in whichsaid host cells are cultured. The antibodies can be isolated from thismedium. Standardized recombinant DNA methods are used in order to obtaingenes for heavy and light antibody chains, to insert said genes intorecombinant expression vectors and to introduce said vectors into hostcells. Methods of this kind are described, for example, in Sambrook,Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al.(eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.

Once DNA fragments encoding VH and VL segments of the antibody ofinterest have been obtained, said DNA fragments may be furthermanipulated using standardized recombinant DNA techniques, for examplein order to convert the genes for variable regions to genes for fulllength antibody chains, to genes for Fab fragments or to an scFv gene.These manipulations comprise linking a VL- or VH-encoding DNA fragmentoperatively to another DNA fragment encoding another protein, forexample a constant antibody region or a flexible linker. The term“operatively linked” is to be understood here as meaning that the twoDNA fragments are linked in such a way that the amino acid sequencesencoded by said two DNA fragments remain in frame.

The isolated DNA encoding the VH region may be converted to a gene for afull length heavy chain by operatively linking the VH-region encodingDNA with another DNA molecule encoding heavy chain constant regions(CH1, CH2 and CH3). The sequences of human heavy chain constant regiongenes are well known (see, for example, Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242),and DNA fragments spanning said regions may be obtained by means ofstandardized PCR amplification. The heavy chain constant region may be aconstant region from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD, withpreference being given to a constant region from IgG, in particular IgG1or IgG4. To obtain a gene for a heavy chain Fab fragment, theVH-encoding DNA may be operatively linked to another DNA moleculeencoding merely the heavy chain constant region CH1.

The isolated DNA encoding the VL region may be converted to a gene for afull length light chain (and a gene for an Fab light chain) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region CL. The sequences of genes of theconstant region of human light chain are well known (see Kabat, E. A.,et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242), and DNA fragments spanning said regions may be obtained bymeans of standardized PCR amplification. The light chain constant regionmay be a constant kappa or lambda region, a constant kappa region beingpreferred.

In order to generate an scFv gene, the VH- and VL-encoding DNA fragmentsmay be operatively linked to another fragment encoding a flexiblelinker, for example the amino acid sequence (Gly₄-Ser)₃ so that the VHand VL sequences are expressed as a continuous single-chain protein,with the VL and VH regions being linked to one another via said flexiblelinker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature(1990) 348:552-554).

Single domain VH and VL having the binding affinities as described abovemay be isolated from single domain libraries by the above-describedmethods. Two VH single-domain chains (with or without CH1) or two VLchains or a pair of one VH chain and one VL chain with the desiredbinding affinity may be useful as described herein for the antibodies ofthe invention.

In order to express the recombinant antibodies or antibody moieties ofthe invention, the DNAs encoding partial or full length light and heavychains may be inserted into expression vectors so as to operatively linkthe genes to appropriate transcriptional and translational controlsequences. In this context, the term “operatively linked” is to beunderstood as meaning that an antibody gene is ligated in a vector insuch a way that transcriptional and translational control sequenceswithin the vector fulfill their intended function of regulatingtranscription and translation of said antibody gene.

Expediently, the expression vector and the expression control sequencesare chosen so as to be compatible with the expression host cell used.The gene for the antibody light chain and the gene for the antibodyheavy chain may be inserted into separate vectors or both genes areinserted into the same expression vector, this being the usual case. Theantibody genes are inserted into the expression vector by means ofstandardized methods (for example by ligation of complementaryrestriction cleavage sites on the antibody gene fragment and the vector,or by ligation of blunt ends, if no restriction cleavage sites arepresent). The expression vector may already carry sequences for antibodyconstant regions prior to insertion of the sequences for the light andheavy chains. For example, one approach is to convert the VH and VLsequences to full length antibody genes by inserting them intoexpression vectors already encoding the heavy and, respectively, lightchain constant regions, thereby operatively linking the VH segment tothe CH segment(s) within the vector and also operatively linking the VLsegment to the CL segment within the vector.

Additionally or alternatively, the recombinant expression vector mayencode a signal peptide which facilitates secretion of the antibodychain from the host cell. The gene for said antibody chain may be clonedinto the vector, thereby linking the signal peptide in frame to the Nterminus of the gene for the antibody chain. The signal peptide may bean immunoglobulin signal peptide or a heterologous signal peptide (i.e.a signal peptide from a non-immunoglobulin protein). In addition to thegenes for the antibody chain, the expression vectors of the inventionmay have regulatory sequences controlling expression of the genes forthe antibody chain in a host cell.

The term “regulatory sequence” is intended to include promoters,enhancers and further expression control elements (e.g. polyadenylationsignals) which control transcription or translation of the genes for theantibody chain. Regulatory sequences of this kind are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). The skilled worker willappreciate that the expression vector design which includes selection ofregulatory sequences may depend on factors such as the choice of thehost cell to be transformed, the desired strength of expression of theprotein, etc. Preferred regulatory sequences for expression in mammalianhost cells include viral elements resulting in strong and constitutiveprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), simian virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus (e.g. the adenovirus major late promoter(AdMLP)) and polyoma. For a further description of viral regulatoryelements and sequences thereof, see, for example, U.S. Pat. No.5,168,062 to Stinski, U.S. Pat. No. 4,510,245 to Bell et a. and U.S.Pat. No. 4,968,615 to Schaffner et al.

Apart from the genes for the antibody chain and the regulatorysequences, the recombinant expression vectors of the invention may haveadditional sequences such as those which regulate replication of thevector in host cells (e.g. origins of replication) and selectable markergenes. The selectable marker genes facilitate the selection of hostcells into which the vector has been introduced (see, for example, U.S.Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all to Axel et al.). Forexample, it is common for the selectable marker gene to render a hostcell into which the vector has been inserted resistant to cytotoxicdrugs such as G418, hygromycin or methotrexate. Preferred selectablemarker genes include the gene for dihydrofolate reductase (DHFR) (foruse in dhfr host cells with methotrexate selection/amplification) andthe neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding said heavy and light chains is(are) transfected into a hostcell by means of standardized techniques. The various forms of the term“transfection” are intended to comprise a multiplicity of techniquescustomarily used for introducing exogenous DNA into a prokaryotic oreukaryotic host cell, for example electroporation, calcium phosphateprecipitation, DEAE-dextran transfection, and the like. Although it istheoretically possible to express the antibodies of the invention eitherin prokaryotic or eukaryotic host cells, preference is given toexpressing the antibodies in eukaryotic cells and, in particular, inmammalian host cells, since the probability of a correctly folded andimmunologically active antibody being assembled and secreted is higherin such eukaryotic cells and in particular mammalian cells than inprokaryotic cells Prokaryotic expression of antibody genes has beenreported as being ineffective for production of high yields of activeantibody (Boss, M. A. and Wood, C R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing recombinant antibodies ofthe invention include CHO cells (including dhfr CHO cells described inUrfaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, whichare used together with a DHFR-selectable marker, as described, forexample, in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenintroducing recombinant expression vectors encoding the antibody genesinto mammalian host cells, the antibodies are produced by culturing thehost cells until the antibody is expressed in said host cells or,preferably, the antibody is secreted into the culture medium in whichthe host cells grow. The antibodies may then be isolated from theculture medium by using standardized protein purification methods.

It is likewise possible to use host cells in order to produce moietiesof intact antibodies, such as Fab fragments or scFv molecules.Variations of the above-described procedure are of course included inthe invention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of the invention. If either light or heavy chainsare present which are not required for binding of the antigen ofinterest, then the DNA encoding either such a light or such a heavychain or both may be removed partially or completely by means ofrecombinant DNA technology. Molecules expressed by such truncated DNAmolecules are likewise included in the antibodies of the invention. Inaddition, it is possible to produce bifunctional antibodies in which aheavy chain and a light chain are an antibody of the invention and theother heavy chain and the other light chain have specificity for anantigen different from the antigen of interest, by crosslinking anantibody of the Invention to a second antibody by means of standardizedchemical methods.

In a preferred system for recombinant expression of an antibody of theinvention or an antigen-binding moiety thereof, a recombinant expressionvector encoding both the antibody heavy chain and the antibody lightchain is introduced into dhfr CHO cells by means of calciumphosphate-mediated transfection. Within the recombinant expressionvector, the genes for the heavy and light antibody chains are in eachcase operatively linked to regulatory CMV enhancer/AdMLP-promoterelements in order to effect strong transcription of said genes. Therecombinant expression vector also carries a DHFR gene which can be usedfor selecting dhfr CHO cells transfected with the vector by usingmethotrexate selection/amplification. The selected transformed hostcells are cultured so that the heavy and light antibody chains areexpressed, and intact antibody is isolated from the culture medium.Standardized molecular-biological techniques are used in order toprepare the recombinant expression vector, to transfect the host cells,to select the transformants, to culture said host cells, and to obtainthe antibody from the culture medium. Thus the invention relates to amethod of synthesizing a recombinant antibody of the invention byculturing a host cell of the invention in a suitable culture mediumuntil a recombinant antibody of the invention has been synthesized. Themethod may furthermore comprise isolating said recombinant antibody fromsaid culture medium.

As an alternative to screening recombinant antibody libraries by phagedisplay, other methods known to the skilled worker may be used forscreening large combinatorial libraries to identify the antibodies ofthe invention. Basically, any expression system in which a closephysical linkage between a nucleic acid and the antibody encoded therebyis established and may be used to select a suitable nucleic acidsequence by virtue of the properties of the antibody it encodes may beemployed.

In one type of an alternative expression system, the recombinantantibody library is expressed in the form of RNA-protein fusions, asdescribed in WO 98/31700 to Szostak and Roberts, and in Roberts, R. W.and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. Inthis system, in-vitro translation of synthetic mRNAs carrying on their3′ end puromycin, a peptidyl acceptor antibiotic, generates a covalentfusion of an mRNA and the peptide or protein encoded by it. Thus aspecific mRNA of a complex mixture of mRNAs (e.g. a combinatoriallibrary) may be concentrated on the basis of the properties of theencoded peptide or protein (e.g. of the antibody or a moiety thereof),such as binding of said antibody or said moiety thereof to Aβ(20-42)globulomer or a derivative thereof. Nucleic acid sequences which encodeantibodies or moieties thereof and which are obtained by screening ofsuch libraries may be expressed by recombinant means in theabove-described manner (e.g. in mammalian host cells) and may, inaddition, be subjected to further affinity maturation by eitherscreening in further rounds mRNA-peptide fusions, introducing mutationsinto the originally selected sequence(s), or using other methods ofin-vitro affinity maturation of recombinant antibodies in theabove-described manner.

Combinations of In-Vivo and In-Vitro Approaches

The antibodies of the invention may likewise be produced by using acombination of in-vivo and in-vitro approaches such as methods in whichAβ(20-42) globulomer or a derivative thereof is first allowed to act onan antibody repertoire in a host animal in vivo to stimulate productionof Aβ(20-42) globulomer- or derivative-binding antibodies and thenfurther antibody selection and/or antibody maturation (i.e.optimization) are accomplished with the aid of one or more in-vitrotechniques. According to one embodiment, a combined method of this kindmay comprise firstly immunizing a nonhuman animal (e.g. a mouse, rat,rabbit, chicken, camelid, sheep or goat or a transgenic version thereofor a chimeric mouse) with said Aβ(20-42) globulomer or derivativethereof to stimulate an antibody response to the antigen and thenpreparing and screening a phage display antibody library by usingimmunoglobulin sequences of lymphocytes which have been stimulated invivo by the action of said Aβ(20-42) globulomer or derivative. The firststep of this combined procedure may be carded out in the mannerdescribed above in connection with the in-vivo approaches, while thesecond step of this procedure may be carried out in the manner describedabove in connection with the in-vitro approaches. Preferred methods ofhyperimmunizing nonhuman animals with subsequent in-vitro screening ofphage display libraries prepared from said stimulated lymphocytesinclude those described by BioSite Inc., see, for example, WO 98/47343,WO 91/17271, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,580,717.

According to another embodiment, a combined method comprises firstlyimmunizing a nonhuman animal (e.g. a mouse, rat, rabbit, chicken,camelid, sheep, goat or a knockout and/or transgenic version thereof, ora chimeric mouse) with an Aβ(20-42) globulomer of the invention orderivative thereof to stimulate an antibody response to said Aβ(20-42)globulomer or derivative thereof and selecting the lymphocytes whichproduce the antibodies having the desired specificity by screeninghybridomas (prepared, for example, from the immunized animals). Thegenes for the antibodies or single domain antibodies are Isolated fromthe selected clones (by means of standardized cloning methods such asreverse transcriptase polymerase chain reaction) and subjected toin-vitro affinity maturation in order to improve thereby the bindingproperties of the selected antibody or the selected antibodies. Thefirst step of this procedure may be conducted in the manner describedabove in connection with the in-vivo approaches, while the second stepof this procedure may be conducted in the manner described above inconnection with the in-vitro approaches, in particular by using methodsof in-vitro affinity maturation, such as those described in WO 97/29131and WO 00/56772.

In a further combined method, the recombinant antibodies are generatedfrom individual isolated lymphocytes by using a procedure which is knownto the skilled worker as selected lymphocyte antibody methods (SLAM) andwhich is described in U.S. Pat. No. 5,627,052, WO 92/02551 and Babcock,J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In thismethod, a non-human animal (e.g. a mouse, rat, rabbit, chicken, camelid,sheep, goat, or a transgenic version thereof, or a chimeric mouse) isfirstly immunized in vivo with Aβ(20-42) globulomer or a derivativethereof to stimulate an immune response to said oligomer or derivative,and then individual cells secreting antibodies of interest are selectedby using an antigen-specific haemolytic plaque assay. To this end, theglobulomer or derivative thereof or structurally related molecules ofinterest may be coupled to sheep erythrocytes, using a linker such asbiotin, thereby making it possible to identify individual cellssecreting antibodies with suitable specificity by using the haemolyticplaque assay. Following the identification of cells secreting antibodiesof interest, cDNAs for the variable regions of the light and heavychains are obtained from the cells by reverse transcriptase PCR, andsaid variable regions may then be expressed in association with suitableimmunoglobulin constant regions (e g. human constant regions) inmammalian host cells such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences derived from invivo-selected lymphocytes may then be subjected to further in-vitroanalysis and in-vitro selection by spreading out the transfected cells,for example, in order to isolate cells expressing antibodies with thebinding affinity. The amplified immunoglobulin sequences may furthermorebe manipulated in vitro.

Antibodies having the required affinities defined herein can be selectedby performing a dot blot essentially as described above. Briefly, theantigen is attached to a solid matrix, preferably dotted onto anitrocellulose membrane, in serial dilutions. The immobilized antigen isthen contacted with the antibody of interest followed by detection ofthe latter by means of an enzyme-conjugated secondary antibody and acolorimetric reaction; at defined antibody and antigen concentrations,the amount of antibody bound allows affinity determination. Thus therelative affinity of two different antibodies to one target, or of oneantibody to two different targets, is here defined as the relation ofthe respective amounts of target-bound antibody observed with the twoantibody-target combinations under otherwise identical dot blotconditions.

Antibodies which bind to the same epitope as monoclonal antibody 5F7,10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1, or 3B10 can be obtained in amanner known per se.

In the same way as antibodies may be competing, described above,different target structures are herein said to be “competing” for aparticular antibody if at least one of these structures is capable ofspecifically reducing the measurable binding of another, preferably byoffering an overlapping or identical epitope, more preferably anidentical epitope.

Competing target entities are useful for directly selecting antibodiesby virtue of their relative affinity to such target structures. Relativeaffinities may thus be determined directly by using a competition assayin which distinguishable forms of the competing entities, e. g.differently labelled competing structures, are contacted with theantibody of interest, and the relative affinity of the antibody to eachof these entities is deduced from the relative amounts of these entitieswhich are bound by the antibody.

Such competition may be used to directly enrich for antibodiespossessing a desired relative affinity to the target entity, byattaching the entity towards which greater affinity is desired to asolid matrix support and adding a suitable amount, preferably a molarexcess, of the competing entity towards which smaller affinity isdesired to the medium. Thus, the antibodies displaying the desiredrelative affinities will tend to bind to the matrix more strongly thanothers and may be obtained after washing out the less desirable forms,e. g. by washing out at low salt concentrations and then harvesting thebound antibody by reversibly detaching it from its target by using highsalt concentrations. If desired, several rounds of enrichment may beperformed. In a particular embodiment of the invention, where thegenotype underlying an antibody is physically linked to this antibody,e. g. in a pool of hybridomas or antigen-displaying phages or yeastcells, the corresponding phenotype may be rescued.

In another embodiment of the invention, a modified dot blot is usedwhere the immobilized antigen competes with a solved entity for antibodybinding, so that the relative affinity of the antibody can be deducedfrom the percentage bound to the immobilized antigen.

Antibody moieties such as Fab and F(ab′)₂ fragments may be produced fromwhole antibodies by using conventional techniques such as digestion withpapain or pepsin. In addition, antibodies, antibody moieties andimmunoadhesion molecules may be obtained by using standardizedrecombinant DNA techniques.

The present invention also relates to pharmaceutical agents(compositions) comprising an antibody of the invention and, optionally,a pharmaceutically suitable carrier. Pharmaceutical compositions of theinvention may furthermore contain at least one additional therapeuticagent, for example one or more additional therapeutic agents for thetreatment of a disease for whose relief the antibodies of the inventionare useful. If, for example, the antibody of the Invention binds to aglobulomer of the invention, the pharmaceutical composition mayfurthermore contain one or more additional therapeutic agents useful forthe treatment of disorders in which the activity of said globulomer isimportant.

Pharmaceutically suitable carriers include any solvents, dispersingmedia, coatings, antibacterial and antifungal agents, isotonic andabsorption-delaying agents, and the like, as long as they arephysiologically compatible. Pharmaceutically acceptable carriersinclude, for example, water, saline, phosphate-buffered saline,dextrose, glycerol, ethanol and the like, and combinations thereof. Inmany cases, preference is given to using isotonic agents, for examplesugars, polyalcohols such as mannitol or sorbitol, or sodium chloride inaddition. Pharmaceutically suitable carriers may furthermore containrelatively small amounts of auxiliary substances such as wetting agentsor emulsifiers, preservatives or buffers, which increase the half lifeor efficacy of the antibodies.

The pharmaceutical compositions may be suitable for parenteraladministration, for example. Here, the antibodies are preparedpreferably as Injectable solutions with an antibody content of 0.1-250mg/ml. The injectable solutions may be prepared in liquid or lyophilizedform, the dosage form being a flint glass or vial, an ampoule or afilled syringe. The buffer may contain L-histidine (1-50 mM, preferably5-10 mM) and have a pH of 5.0-7.0, preferably of 6.0. Further suitablebuffers include, without being limited thereto, sodium succinate, sodiumcitrate, sodium phosphate or potassium phosphate buffers. Sodiumchloride may be used in order to adjust the tonicity of the solution toa concentration of 0-300 mM (preferably 150 mM for a liquid dosageform). Cryoprotectants, for example sucrose (e g. 0-10%, preferably0.5-1.0%) may also be included for a lyophilized dosage form. Othersuitable cryoprotectants are trehalose and lactose. Fillers, for examplemannitol (e.g. 1-10%, preferably 2-4%) may also be included for alyophilized dosage form. Stabilizers, for example L-methionine (e.g.51-50 mM, preferably 5-10 mM) may be used both in liquid and lyophilizeddosage forms. Further suitable fillers are glycine and arginine.Surfactants, for example polysorbate 80 (e. g. 0-0.05%, preferably0.005-0.01%), may also be used. Further surfactants are polysorbate 20and BRIJ surfactants.

The compositions of the invention may have a multiplicity of forms.These Include liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g. injectable and Infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended type of administration and on thetherapeutic application. Typically, preference is given to compositionsin the form of injectable or infusible solutions, for examplecompositions which are similar to other antibodies for passiveimmunization of humans. The preferred route of administration isparenteral (e.g. intravenous, subcutaneous, intraperitoneal orintramuscular). According to a preferred embodiment, the antibody isadministered by intravenous infusion or injection. According to anotherpreferred embodiment, the antibody is administered by Intramuscular orsubcutaneous injection.

Therapeutic compositions must typically be sterile and stable underpreparation and storage conditions. The compositions may be formulatedas solutions, microemulsions, dispersions, liposomes or other orderedstructures suitable for high concentrations of active substance. Sterileinjectable solutions may be prepared by introducing the active compound(i.e. the antibody) in the required amount into a suitable solvent,where appropriate with one or a combination of the abovementionedingredients, as required, and then sterile-filtering said solution.Dispersions are usually prepared by introducing the active compound intoa sterile vehicle containing a basic dispersion medium and, whereappropriate, other required ingredients. In the case of a sterilelyophilized powder for preparing sterile injectable solutions, vacuumdrying and spray drying are preferred methods of preparation, whichproduces a powder of the active ingredient and, where appropriate, offurther desired ingredients from a previously sterile-filtered solution.The correct flowability of a solution may be maintained by using, forexample, a coating such as lecithin, by maintaining, in the case ofdispersions the required particle size or by using surfactants. Aprolonged absorption of injectable compositions may be achieved byadditionally introducing into the composition an agent which delaysabsorption, for example monostearate salts and gelatine.

The antibodies of the invention may be administered by a multiplicity ofmethods known to the skilled worker, although the preferred type ofadministration for many therapeutic applications is subcutaneousinjection, Intravenous injection or infusion. The skilled worker willappreciate that the route and/or type of administration depend on theresult desired According to particular embodiments, the active compoundmay be prepared with a carrier which protects the compound against rapidrelease, such as, for example, a formulation with sustained orcontrolled release, which includes implants, transdermal plasters andmicroencapsulated release systems. Biologically degradable biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters and polylactic acid may be used. Themethods of preparing such formulations are well known to the skilledworker; see, for example, Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

According to particular embodiments, an antibody of the invention may beadministered orally, for example in an inert diluent or a metabolizableedible carrier. The antibody (and further ingredients, if desired) mayalso be enclosed in a hard or soft gelatine capsule, compressed totablets or added directly to food. For oral therapeutic administration,the antibodies may be mixed with excipients and used in the form of oraltablets, buccal tablets, capsules, elixirs, suspensions, syrups and thelike. If it is intended to administer an antibody of the invention via aroute other than the parenteral one, it may be necessary to choose acoating from a material which prevents its inactivation.

The present invention also relates to a method of inhibiting theactivity of globulomers of the invention in an individual which suffersfrom a disorder in which the amyloid β protein is involved and in whichin particular the activity of said globulomers of the invention isimportant Said method comprises the administration of at least oneantibody of the invention to the individual with the aim of inhibitingthe activity of the globulomer to which the antibody binds. Saidindividual is preferably a human being. An antibody of the invention maybe administered for therapeutic purposes to a human individual. Inaddition, an antibody of the invention may be administered to a nonhumanmammal for veterinary purposes or within the framework of an animalmodel for a particular disorder. Such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the invention (forexample for testing dosages and time courses of administration).

Disorders in which the globulomers of the invention play a part includein particular disorders in whose development and/or progression aglobulomer of the invention is involved. These are in particular thosedisorders in which globulomers of the invention are evidently orpresumably responsible for the pathophysiology of said disorder or are afactor which contributes to the development and/or progression of saiddisorder. Accordingly, those disorders are included here in whichinhibition of the activity of globulomers of the Invention can relievesymptoms and/or progression of the disorder. Such disorders can beverified, for example, by an increased concentration of globulomers ofthe invention in a biological fluid of an individual suffering from aparticular disorder (e g. Increased concentration in serum, plasma, CSF,urine, etc.). This may be detected, for example, by using an antibody ofthe invention. The globulomers of the invention play an important partin the pathology associated with a multiplicity of disorders in whichneurodegenerative elements, cognitive deficiencies, neurotoxic elementsand inflammatory elements are involved.

In another aspect of the Invention, disorders that can be treated orprevented include those associated with amyloidoses. The term“amyloidoses” here denotes a number of disorders characterized byabnormal folding, dumping, aggregation and/or accumulation of particularproteins (amyloids, fibrous proteins and their precursors) in varioustissues of the body. In Alzheimer's disease and Down's syndrome, nervetissue is affected, and in cerebral amyloid angiopathy (CAA) bloodvessels are affected.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody moiety of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody moiety may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodymoiety to elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the antibody or antibody portion are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Moreover, the present invention includes a further method of preventingor treating Alzheimer's disease in a patient in need of such preventionor treatment. This method comprises the step of administering thevaccine noted above to the patient in an amount sufficient to effect theprevention or treatment. Further, the present invention encompasses amethod of identifying compounds suitable for active immunization of apatient predicted to develop an amyloidosis, e.g. Alzheimer's disease.

This method comprises: 1) exposing one or more compounds of interest toone or more of the antibodies described above for a time and underconditions sufficient for the one or more compounds to bind to theantibody or antibodies; 2) identifying those compounds which bind to theantibody or antibodies, the identified compounds to be used in activeimmunization in a patient predicated to develop an amyloidosis, e.g.Alzheimer's disease.

Within the framework of diagnostic usage of the antibodies, qualitativeor quantitative specific globulomer determination serves in particularto diagnose disease-relevant amyloid β forms In this context,specificity means the possibility of being able to detect a particularglobulomer or a derivative thereof, or a mixture thereof with sufficientsensitivity. The antibodies of the invention advantageously havedetection threshold concentrations of less than 10 ng/ml of sample,preferably of less than 1 ng/ml of sample and particularly preferably ofless than 100 pg/ml of sample, meaning that at least the concentrationof globulomer per ml of sample, indicated in each case, advantageouslyalso lower concentrations, can be detected by the antibodies of theinvention.

The detection is carried out immunologically. This may be carried out inprinciple by using any analytical or diagnostic assay method in whichantibodies are used, including agglutination and precipitationtechniques, immunoassays, immunohistochemical methods and immunoblottechniques, for example Western blotting or, preferably, dot blotmethods. In vivo methods, for example imaging methods, are also includedhere.

The use in immunoassays is advantageous. Competitive immunoassays, i.e.assays where antigen and labelled antigen (tracer) compete for antibodybinding, and sandwich immunoassays, i.e. assays where binding ofspecific antibodies to the antigen is detected by a second, usuallylabelled antibody, are both suitable. These assays may be eitherhomogeneous, i.e. without separation into solid and liquid phases, orheterogeneous, i.e. bound labels are separated from unbound ones, forexample via solid phase-bound antibodies. Depending on labelling andmethod of measurement, the various heterogeneous and homogeneousimmunoassay formats can be classified into particular classes, forexample RIAs (radioimmunoassays), ELISA (enzyme-linked Immunosorbentassay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay),TRFIA (time-resolved FIA), IMAC (immunoactivation), EMIT(enzyme-multiplied immune test), TIA (turbidometric immunoassay), I-PCR(immuno-PCR).

For the globulomer quantification of the invention, preference is givento competitive immunoassays in which a defined amount of labelledglobulomer derivative serving as tracer competes with the globulomer ofthe sample (containing an unknown amount of unlabelled globulomers) tobe quantified for binding to the antibody used. The amount of antigen,i.e. the amount of globulomer, in the sample can be determined from theamount of the displaced tracer with the aid of a standard curve.

Of the labels available for these purposes, enzymes have provedadvantageous. Systems based on peroxidases, in particular horseradishperoxidase, alkaline phosphatase and β-D-galactosidase, may be used, forexample. Specific substrates whose conversion can be monitoredphotometrically, for example, are available for these enzymes. Suitablesubstrate systems are based on p-nitrophenyl phosphate (p-NPP),5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NPT),Fast-Red/naphthol-AS-TS phosphate for alkaline phosphatase;2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPT), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 3-dimethylaminobenzoic acid (DMAB)and 3-methyl-2-benzothiazolinehydrazone (MBTH) for peroxidases;o-nitrophenyl-β-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and4-methylumbelliphenyl-β-D-galactoside (MUG) for β-D-galactosidase. Inmany cases, these substrate systems are commercially available in aready-to-use form, for example in the form of tablets which may alsocontain further reagents such as appropriate buffers and the like.

The tracers used may be labelled globulomers. In this sense, aparticular globulomer can be determined by labelling the globulomer tobe determined and using it as tracer.

The coupling of labels to globulomers for preparing tracers may becarried out in a manner known per se. The comments above onderivatization of globulomers of the invention are referred to byanalogy. In addition, a number of labels appropriately modified forconjugation to proteins are available, for example biotin-, avidin-,extravidin- or streptavidin-conjugated enzymes, maleimide-activatedenzymes and the like. These labels may be reacted directly with theoligomer or, if required, with the appropriately derivatized globulomerto give the tracer. If, for example, a streptavidin-peroxidase conjugateis used, then this firstly requires biotinylation of the globulomer.This applies correspondingly to the reverse order. Suitable methods tothis end are also known to the skilled worker.

If a heterogeneous immunoassay format is chosen, the antigen-antibodycomplex may be separated by binding it to the support, for example viaan anti-idiotypical antibody coupled to said support, e.g. an antibodydirected against rabbit IgG. Appropriate supports, in particularmicrotiter plates coated with appropriate antibodies, are known andpartly commercially available.

The present invention further relates to immunoassay sets having atleast one antibody as described above and further components. Said setsare, usually in the form of a packaging unit, a combination of means forcarrying out a globulomer determination of the invention. For thepurpose of as easy handling as possible, said means are preferablyprovided in an essentially ready-to-use form. An advantageousarrangement offers the immunoassay in the form of a kit. A kit usuallycomprises multiple containers for separate arrangement of components.All components may be provided in a ready-to-use dilution, as aconcentrate for diluting or as a dry substance or lyophilisate fordissolving or suspending; individual or all components may be frozen orstored at room temperature until use. Sera are preferably shock-frozen,for example at −20° C. so that in these cases an immunoassay has to bekept preferably at temperatures below freezing prior to use.

Further components supplied with the immunoassay depend on the type ofsaid immunoassay. Usually, standard protein, tracer which may or may notbe required and control serum are supplied together with the antiserum.Furthermore, microtiter plates, preferably antibody-coated, buffers, forexample for testing, for washing or for conversion of the substrate, andthe enzyme substrate itself may also be included.

General principles of immunoassays and generation and use of antibodiesas auxiliaries in laboratory and hospital can be found, for example, inAntibodies, A Laboratory Manual (Harlow, E., and Lane, D., Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).

Thus, the present invention also includes a method of diagnosing anamyloidosis, e.g. Alzheimer's disease, in a patient suspected of havingthis disease. This method comprises the steps of: 1) Isolating abiological sample from the patient; 2) contacting the biological samplewith at least one of the antibodies described above for a time and underconditions sufficient for formation of antigen/antibody complexes; and3) detecting presence of the antigen/antibody complexes in said sample,presence of the complexes indicating a diagnosis of an amyloidosis, e.g.Alzheimer's disease, in the patient. The antigen may be, for example, anglobulomer or a portion or fragment thereof which has the samefunctional properties as the full globulomer (e.g., binding activity).

Further, the present invention includes another method of diagnosing anamyloidosis, e.g. Alzheimer's disease in a patient suspected of havingthis disease. This method comprising the steps of: 1) isolating abiological sample from the patient; 2) contacting the biological samplewith an antigen for a time and under conditions sufficient for theformation of antibody/antigen complexes; 3) adding a conjugate to theresulting antibodylantigen complexes for a time and under conditionssufficient to allow the conjugate to bind to the bound antibody, whereinthe conjugate comprises one of the antibodies described above, attachedto a signal generating compound capable of generating a detectablesignal; and 4) detecting the presence of an antibody which may bepresent in the biological sample, by detecting a signal generated by thesignal generating compound, the signal indicating a diagnosis of anamyloidosis, e.g. Alzheimer's disease in the patient. The antigen may bea globulomer or a portion or fragment thereof having the same functionalproperties as the full globulomer (e.g., binding activity).

The present invention includes an additional method of diagnosing anamyloidosis, e.g. Alzheimer's disease in a patient suspected of havingan amyloidosis, e g. Alzheimer's disease. This method comprises thesteps of: 1) isolating a biological sample from said patient; 2)contacting the biological sample with anti-antibody, wherein theanti-antibody is specific for one of the antibodies described above, fora time and under conditions sufficient to allow for formation ofanti-antibody/antibody complexes, the complexes containing antibodypresent in the biological sample; 2) adding a conjugate to resultinganti-antibody/antibody complexes for a time and under conditionssufficient to allow the conjugate to bind to bound antibody, wherein theconjugate comprises an antigen, which binds to a signal generatingcompound capable of generating a detectable signal; and 3) detecting asignal generated by the signal generating compound, the signalindicating a diagnosis of an amyloidosis, e.g. Alzheimer's disease inthe patient.

Also, the present invention includes a kit comprising: a) at least oneof the antibodies described above and b) a conjugate comprising anantibody attached to a signal-generating compound, wherein the antibodyof the conjugate is different from the isolated antibody.

The present invention also encompasses a kit comprising: a) ananti-antibody to one of the antibodies described above and b) aconjugate comprising an antigen attached to a signal-generatingcompound. The antigen may be a globulomer or a fragment or portionthereof having the same functional characteristics as the globulomer(e.g., binding activity).

In one diagnostic embodiment of the present invention, an antibody ofthe present invention, or a portion thereof, is coated on a solid phase(or is present in a liquid phase). The test or biological sample (e.g.,whole blood, cerebrospinal fluid, serum, etc.) is then contacted withthe solid phase. If antigen (e.g., globulomer) is present in the sample,such antigens bind to the antibodies on the solid phase and are thendetected by either a direct or indirect method. The direct methodcomprises simply detecting presence of the complex itself and thuspresence of the antigens. In the indirect method, a conjugate is addedto the bound antigen. The conjugate comprises a second antibody, whichbinds to the bound antigen, attached to a signal-generating compound orlabel. Should the second antibody bind to the bound antigen, thesignal-generating compound generates a measurable signal. Such signalthen indicates presence of the antigen in the test sample.

Examples of solid phases used in diagnostic immunoassays are porous andnon-porous materials, latex particles, magnetic particles,microparticles (see U.S. Pat. No. 5,705,330), beads, membranes,microtiter wells and plastic tubes. The choice of solid phase materialand method of labeling the antigen or antibody present in the conjugate,if desired, are determined based upon desired assay format performancecharacteristics.

As noted above, the conjugate (or indicator reagent) will comprise anantibody (or perhaps anti-antibody, depending upon the assay), attachedto a signal-generating compound or label. This signal-generatingcompound or “label” is itself detectable or may be reacted with one ormore additional compounds to generate a detectable product. Examples ofsignal-generating compounds include chromogens, radioisotopes (e.g.,125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e g.,acridinium), particles (visible or fluorescent), nucleic acids,complexing agents, or catalysts such as enzymes (e.g., alkalinephosphatase, acid phosphatase, horseradish peroxidase,beta-galactosidase and ribonuclease). In the case of enzyme use (e.g.,alkaline phosphatase or horseradish peroxidase), addition of a chromo-,fluro-, or lumo-genic substrate results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, amplification (e.g., polymerase chainreaction) and Raman spectroscopy are also useful.

Examples of biological fluids which may be tested by the aboveimmunoassays include plasma, whole blood, dried whole blood, serum,cerebrospinal fluid or aqueous or organo-aqueous extracts of tissues andcells.

The present invention also encompasses a method for detecting thepresence of antibodies in a test sample. This method comprises the stepsof: (a) contacting the test sample suspected of containing antibodieswith anti-antibody specific for the antibodies in the patient sampleunder time and conditions sufficient to allow the formation ofanti-antibody/antibody complexes, wherein the anti-antibody is anantibody of the present invention which binds to an antibody in thepatient sample; (b) adding a conjugate to the resultinganti-antibody/antibody complexes, the conjugate comprising an antigen(which binds to the anti-antibody) attached to a signal generatingcompound capable of detecting a detectable signal; and (d) detecting thepresence of the antibodies which may be present in the test sample bydetecting the signal generated by the signal generating compound. Acontrol or calibrator may be used which comprises antibody to theanti-antibody.

Kits are also included within the scope of the present invention. Morespecifically, the present invention includes kits for determining thepresence of antigens (e.g., globulomers) in a patient suspected ofhaving Alzheimer's disease or another condition characterized bycognitive impairment. In particular, a kit for determining the presenceof antigens in a test sample comprises a) an antibody as defined hereinor moiety thereof; and b) a conjugate comprising a second antibody(having specificity for the antigen) attached to a signal generatingcompound capable of generating a detectable signal. The kit may alsocontain a control or calibrator which comprises a reagent which binds tothe antigen.

The present invention also includes a kit for detecting antibodies in atest sample. The kit may comprise a) an anti-antibody specific (forexample, one of the subject invention) for the antibody of interest, andb) an antigen or portion thereof as defined above. A control orcalibrator comprising a reagent which binds to the antigen may also beincluded. More specifically, the kit may comprise a) an anti-antibody(such as the one of the present invention) specific for the antibody andb) a conjugate comprising an antigen (e.g., globulomer) attached to asignal generating compound capable of generating a detectable signal.Again, the kit may also comprise a control of calibrator comprising areagent which binds to the antigen.

The kit may also comprise one container such as vial, bottles or strip,with each container with a pre-set solid phase, and other containerscontaining the respective conjugates. These kits may also contain vialsor containers of other reagents needed for performing the assay, such aswashing, processing and indicator reagents.

It should also be noted that the subject invention not only includes thefull length antibodies described above but also moieties or fragmentsthereof, for example, the Fab portion thereof. Additionally, the subjectinvention encompasses any antibody having the same properties of thepresent antibodies in terms of, for example, binding specificity,structure, etc.

Advantages of the Invention

By immunization with Aβ(20-42) globulomer different monoclonalantibodies may be obtained which differ in their tolerance orrecognition of different Aβ(1-42) oligomers and Aβ(X-42) oligomers, asdetermined by comparative dot blotting as described above. This allowsdevelopment of an antibody directed to N-terminally truncated Aβoligomers which possesses an optimal relation between cognitionenhancing effect, desired specificity over other Aβ forms and minimalside effect profile. Surprisingly, the Aβ(1-42) and Aβ(12-42)globulomers, in spite of containing the structural element, are onlypartly recognized by antibodies obtained by using the further truncatedAβ(20-42) globulomer as antigen.

By restriction of the specific oligomeric Aβ form to the basicstructural principle (i.e. use of the N-terminally truncated Aβglobulomers) an antibody profile is generated in active immunizationwhich is highly specific for oligomeric Aβ forms. The same holds truefor monoclonal antibodies for use in passive immunization. The advantageof such a specific strategy for immunization (active and passive) isthat it will not induce an immune response against Aβ monomers, Aβpeptides in fibrillary states of aggregation or sAPPα. This isadvantageous in several ways:

-   1) In the form of insoluble Aβ plaques Aβ peptides in fibrillary    states of aggregation amount to the major part of the entire Aβ    peptide pool in AD brains. A massive release of Aβ by dissolution of    Aβ plaques induced by reaction of anti-Aβ antibodies with these    plaques is to be regarded as detrimental. This massive release of Aβ    would then cross the blood-brain barrier, enter the bloodstream and    potentially increase the risk of microhaemorrhages. In addition, in    the ELAN trial this very strategy of immunization with fibrillary Aβ    peptide forms required cancellation of the trial due to 6% of cases    with an onset of meningoencephalitis.-   2) Immune responses directed to monomeric Aβ peptide forms are    undesirable, as it was possible to show that the latter may exert    cognition-enhancing effects.-   3) Immune responses directed to sAPPα are likewise undesirable, as    this might lead to a reaction with the physiologically occurring    precursor protein APP and thus to an auto-immune reaction. Moreover,    sAPPα was also shown to exert cognition-enhancing effects.-   4) A response directed to vascular Aβ peptide in the form of CAA is    to be avoided in order to eschew the undesirable side effect of    microhaemorrhages (antibodies against the central portion of Aβ and    which in addition do not bind to AP-peptides aggregated in the form    of CAA induce fewer microhaemorrhages when compared to such against    the N-terminus, see above).-   5) Antibodies which specifically react with Aβ oligomers will have    higher bioavailability with regard to the pathophysiologically    relevant Aβ species, as they will not be bound to, e.g., fibrillary    Aβ and thus made unavailable for therapeutic effect.

Deposit Information: The hybridoma which produces monoclonal antibody5F7 was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110 on Dec. 1, 2005 under theterms of the Budapest Treaty and received designation PTA-7241. Further,the hybridoma which produces monoclonal antibody 10F11 was depositedwith the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 10801 on Dec. 1, 2005 under the terms of the BudapestTreaty and received designation PTA-7239. Additionally, the hybridomawhich produces monoclonal antibody 4B7 was deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va. 10801on Dec. 1, 2005 under the terms of the Budapest Treaty and receiveddesignation PTA-7242, and the hybridoma which produces monoclonalantibody 7C6 was deposited with the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 10801 on Dec. 1, 2005 underthe terms of the Budapest Treaty and received designation PTA-7240.Additionally, the hybridoma which produces monoclonal antibody 6A2 wasdeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 10801 on Feb. 28, 2006 under the terms of theBudapest Treaty and received designation PTA-7409, and the hybridomawhich produces monoclonal antibody 2F2 was deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va. 10801on Feb. 28, 2006 under the terms of the Budapest Treaty and receiveddesignation PTA-7408 The hybridoma which produces monoclonal antibody4010 was deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 10801 on Feb. 28, 2006 under theterms of the Budapest Treaty and received designation PTA-7405. Thehybridoma which produces monoclonal antibody 7E5 was deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 10801 on Aug. 16, 2006 under the terms of the Budapest Treaty andreceived designation PTA-7809. The hybridoma which produces monoclonalantibody 10C1 was deposited with the American Type Culture Collection,10801 University Boulevard, Manassas, Va. 10801 on Aug. 16, 2006 underthe terms of the Budapest Treaty and received designation PTA-7810. Thehybridoma which produces monoclonal antibody 3B10 was deposited with theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 10801 on Sep. 1, 2006 under the terms of the Budapest Treaty andreceived designation PTA-7851. A1 deposits have been made on behalf ofAbbott Laboratories, 100 Abbott Park Road, Abbott Park, Ill. 60064 (US).

In the drawings:

FIG. 1 shows size-exclusion chromatograms of Aβ(1-42) and Aβ(1-40).Aβ(1-42) monomer was dissolved in A) 0.1% NH₄OH, B) 70% formic acid C)0.1% NaOH and in D) Aβ(1-40) was dissolved in 0.1% NaOH. Subsequently,the samples were further diluted 1:10 in 20 mM NaH₂PO₄, 140 mM NaCl, pH7.4 These samples were incubated for 5 min (left column) or 1 hour(right column) after dissolution at ambient temperature, then applied tothe size exclusion column;

FIG. 2 A) shows an SDS PAGE of standard proteins (molecular markerproteins, lane 1); Aβ(1-42) fibril preparation; control (lane 2);Aβ(1-42) fibril preparation+mAb 5F7, 20 h, 37° C., supernatant (lane 3);Aβ(1-42) fibril preparation+mAb 5F7, 20 h, 37° C., pellet (lane 4);Aβ(1-42) fibril preparation+mAb 6E10, 20 h, 37° C., supernatant (lane5); Aβ(1-42) fibril preparation+mAb 6E10, 20 h 37° C., pellet (lane6););

-   -   B) shows the results of the quantitative analysis of mAbs bound        to Aβ-fibrils in percent of total antibody;

FIG. 3 is a bar diagram which shows the results of the objectrecognition test with APP/L transgenic mice after active immunizationwith Aβ(1-42) monomers in 0.1% NH₄OH, Aβ(1-42) globulomers and Aβ(20-42)globulomers as compared to wild-type mice (positive control) andPBS-treated APP/L mice (negative control), where circles indicatesignificant differences to PBS-treated APP/L mice and asterisks indicatehighly significant differences to chance level (50%) according topost-hoc t-test after P<0.05 in ANOVA for differences among groups;

FIG. 4 shows dot blots of the reactivity of 100 pmol/μl (row A); 10pmol/μl (row B); 1 pmol/μl (row C), 0.1 pmol/μl (row D) and 0.01 pmol/μl(row E) of Aβ(1-42) globulomer (column 1), of HFIP pretreated Aβ(1-42)monomer in Pluronic F68 (column 2), of Aβ(20-42) globulomer (column 3),of Aβ(12-42) globulomer (column 4); of HFIP pretreated Aβ(1-40) monomerin DMSO (column 5); of Aβ(1-42) monomer, NH₄OH (column 6); of anAβ(1-42) fibril preparation (column 7); and of sAPPα from Sigma (column8) with various antisera obtained after an active immunization of APP/ILTg mice with Aβ(20-42) globulomer;

FIG. 5 is a bar diagram which shows the concentrations of soluble andInsoluble Aβ(1-42) and Aβ(1-40) peptide in brain extracts of activelyimmunized APP/PS1 Tg-mice with either Aβ(1-42) monomer (0.1% NH₄OH),Aβ(1-42) globulomer, Aβ(20-42) globulomer or vehicle as control;

FIG. 6 is a bar diagram which shows the results of the objectrecognition test with APP/L transgenic mice after passive immunizationwith anti Aβ(20-42) globulomer antibodies 5F7, 10F11, and 7C6 ascompared to control mice for A) each antibody separately and B) for allantibodies taken together;

FIG. 7 A) shows a dot blot analysis of the specificity of differentanti-Aβ antibodies (−6E10, −5F7, −4B7, −10F11, −6A2, −4D10, −3B10, −2F2,−7C6, −7E5, −10C1). The monoclonal antibodies tested here were obtainedby active immunization of mice with Aβ(20-42) globulomer followed byselection of the fused hybridoma cells (except for the commercialavailable 6E10, Signet No 9320). The individual Aβ forms were applied inserial dilutions and incubated with the respective monoclonal antibodiesfor immune reaction:

-   -   1. Aβ(1-42) monomer, 0.1% NH₄OH    -   2. Aβ(1-40) monomer, 0.1% NH₄OH    -   3. Aβ(1-42) monomer, 0.1% NaOH    -   4. Aβ(1-40) monomer, 0.1% NaOH    -   5. Aβ(1-42) globulomer    -   6. Aβ(12-42) globulomer    -   7. Aβ(20-42) globulomer    -   8. Aβ(1-42) fibril preparation    -   9. sAPPα (Sigma) (first dot: 1 pmol)    -   B) Quantitative evaluation was done using a densitometric        analysis of the intensity. For each Aβ form, only the dot        corresponding to the lowest antigen concentration was evaluated        provided that it had a relative density of greater than 20% of        the relative density of the last optically unambiguously        Identified dot of the Aβ(20-42) globulomer (threshold). This        threshold value was determined for every dot-blot independently.        The value indicates the relation between recognition of        Aβ(20-42) globulomer and the respective Aβ form for the antibody        given;

FIG. 8 shows the binding of antibodies at different concentrations totransversal sections of the neocortices of Alzheimer's disease (AD)patients or old APP transgenic mice:

-   -   A) Verification of amyloid deposits by Congo Red staining as        plaques in brain tissue and as cerebral amyloid angiopathy (CAA)        in brain vessels in the APP transgenic mouse line Tg2576 and in        an AD patient (RZ55);    -   B) Strong staining of parenchymal deposits of Aβ(amyloid        plaques) in an AD patient (RZ16) occurs only with 6G1 and the        commercially available antibody 6E10 (left column) while        antibodies 5F7, 2F2, and 6A2 (second column), 4D10, 10F11, and        3B10 (third column) and 7C6, 7E5, and 10C1 (right column) show        no staining. All antibodies were used at a concentration of 0.7        μg/ml;    -   C) Strong staining of parenchymal deposits of Aβ(amyloid        plaques) in 19 month old Tg2576 occurs only with 6G1 and the        commercially available antibody 6E10 (left column) while        antibodies 5F7, 2F2, and 6A2 (second column), 4D10, 10F11, and        3B10 (third column) and 7C6, 7E5, and 10C1 (right column) show        no staining. All antibodies were used at a concentration of 0.7        μg/ml;    -   D)-G) Quantification of the analysis of Aβ plaque staining in        the histological images using image analysis. Optical density        values (0%=no staining) were calculated from the greyscale        values of plaques subtracted by greyscale values of background        tissue: D) Staining at 0.7 μg/ml antibody in old Tg2576 mice, E)        staining at 3 different concentrations of antibodies in APP/L        mice, F) staining at 0.7 μg/ml antibody in an AD patient (RZ55),        and G) staining at 3 different concentrations of antibodies in        an AD patient (RZ16). The differences between staining of the        commercially available antibodies 6E10 (asterisks) and 4G8        (circles) and all other antibodies (three asterisks/circles:        p<0.001 versus control; post-hoc Bonferroni's t-test after ANOVA        with p<0.001) were statistically evaluated (D, F). In E) and G)        all antibodies except 6G1 showed always significantly less        staining than the commercially available antibodies 6E10 and 4G8        (p<0.001 in post-hoc t-test after p<0.001 In ANOVA).    -   H) Strong staining of vascular deposits of Aβ (arrows) occurs        only with 6G1 and the commercially available antibody 6E10 (left        column) while antibodies 5F7, 2F2, and 6A2 (second column),        4D10, 10F11, and 3B10 (third column) and 7C6, 7E5, and 10C1        (right column) show no staining. All antibodies were used at a        concentration of 0.7 μg/ml. A qualitatively similar situation        was found in Tg2576 mice (not shown here);

FIG. 9 Anti-Aβ-antibody titer and dot-blot selectivity profile in plasmaof TG2576 mice approximately one year after active immunization. Plasmasamples of Tg2576-mice approximately one year after the lastimmunization with A) Aβ (20-42) globulomer, B) Aβ (12-42) globulomer, C)Aβ (1-42) monomer and D) vehicle, were assessed for anti-Aβ antibodiesproduced and still present by dot-blot.

-   -   1. Aβ (1-42) globulomer    -   2. Aβ (1-42) monomer, HFIP pretreated, in 0.1% Pluronic F68    -   3. Aβ (20-42) globulomer    -   4. Aβ (12-42) globulomer    -   5. Aβ (1-40) monomer, HFIP pre-treated, 5 mM in DMSO    -   6. Aβ (1-42) monomer, 0.1% NH₄OH    -   7. Aβ (1-42) fibril preparation    -   8. sAPPα (Sigma); (first dot: 1 pmol);

FIG. 10 shows a table summarizing the levels of Aβ(20-42) globulomer inbrain tissue of human beings having Alzheimer's disease and a nondemented control;

FIG. 11 illustrates nucleotide and amino acid sequences of the variableheavy and light chains of monoclonal antibodies (mAbs) as follows(complementarity determining regions (CDRs) are underlined in each aminoacid sequence):

FIG. 11 SEQ ID NO: Sequence Type Chain mAb A1 1 nucleotide variableheavy (VH) 5F7 A2 2 nucleotide variable light (VL) 5F7 A1 3 amino acidvariable heavy (VH) 5F7 A2 4 amino acid variable light (VL) 5F7 B1 5nucleotide variable heavy (VH) 10F11 B2 6 nucleotide variable light (VL)10F11 B1 7 amino acid variable heavy (VH) 10F11 B2 8 amino acid variablelight (VL) 10F11 C1 9 nucleotide variable heavy (VH) 7C6 C2 10nucleotide variable light (VL) 7C6 C1 11 amino acid variable heavy (VH)7C6 C2 12 amino acid variable light (VL) 7C6 D1 13 nucleotide variableheavy (VH) 4B7 D2 14 nucleotide variable light (VL) 4B7 D1 15 amino acidvariable heavy (VH) 4B7 D2 16 amino acid variable light (VL) 4B7 E1 17nucleotide variable heavy (VH) 2F2 E2 18 nucleotide variable light (VL)2F2 E1 19 amino acid variable heavy (VH) 2F2 E2 20 amino acid variablelight (VL) 2F2 F1 21 nucleotide variable heavy (VH) 6A2 F2 22 nucleotidevariable light (VL) 6A2 F1 23 amino acid variable heavy (VH) 6A2 F2 24amino acid variable light (VL) 6A2 G1 25 nucleotide variable heavy (VH)4D10 G2 26 nucleotide variable light (VL) 4D10 G1 27 amino acid variableheavy (VH) 4D10 G2 28 amino acid variable light (VL) 4D10 H1 29nucleotide variable heavy (VH) 7E5 H2 30 nucleotide variable light (VL)7E5 H1 31 amino acid variable heavy (VH) 7E5 H2 32 amino acid variablelight (VL) 7E5 I1 33 nucleotide variable heavy (VH) 10C1 I2 34nucleotide variable light (VL) 10C1 I1 35 amino acid variable heavy (VH)10C1 I2 36 amino acid variable light (VL) 10C1 J1 37 nucleotide variableheavy (VH) 3B10 J1 38 amino acid variable heavy (VH) 3B10

The following examples are intended to illustrate the invention, withoutlimiting its scope

Example 1 Preparation of Globulomers a) Aβ(1-42) Globulomer

The Aβ(1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland)was suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propano (HFIP) at 6 mg/mLand incubated for complete solubilization under shaking at 37° C. for1.5 h. The HFIP acts as a hydrogen-bond breaker and is used to eliminatepre-existing structural inhomogeneities in the Aβ peptide. HFIP wasremoved by evaporation in a SpeedVac and Aβ(1-42) resuspended at aconcentration of 5 mM in dimethylsulfoxide and sonicated for 20 s. TheHFIP-pre-treated Aβ(1-42) was diluted in phosphate-buffered saline (PBS)(20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume 2% sodiumdodecyl sulfate (SDS) (in H₂O) added (final concentration of 0.2% SDS).An incubation for 6 h at 37° C. resulted in the 16/20-kDa Aβ(1-42)globulomer (short form for globular oligomer) intermediate. The38/48-kDa Aβ(1-42) globulomer was generated by a further dilution withthree volumes of H₂O and incubation for 18 h at 37° C. Aftercentrifugation at 3000 g for 20 min the sample was concentrated byultrafiltration (30-kDa cut-off), dialysed against 5 mM NaH₂PO₄, 35 mMNaCl, pH 7.4, centrifuged at 10000 g for 10 min and the supernatantcomprising the 38/48-kDa Aβ(1-42) globulomer withdrawn. As analternative to dialysis the 38/48-kDa Aβ(1-42) globulomer could also beprecipitated by a ninefold excess (v/v) of ice-cold methanol/acetic acidsolution (33% methanol, 4% acetic acid) for 1 h at 4° C. The 38/48-kDaAβ(1-42) globulomer is then pelleted (10 min at 16200 g), resuspended in5 mM NaH₂PO₄, 35 mM NaCl, pH 7.4, and the pH adjusted to 7.4.

b) Cross-Linked Aβ(1-42) Globulomer

The Aβ(1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland)was suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6mg/mL and incubated for complete solubilization under shaking at 37° C.for 1.5 h. The HFIP acts as a hydrogen-bond breaker and was used toeliminate pre-existing structural inhomogeneities in the Aβ peptide.HFIP was removed by evaporation in a SpeedVac and Aβ(1-42) resuspendedat a concentration of 5 mM in dimethylsulfoxide and sonicated for 20 s.The HFIP-pre-treated Aβ(1-42) was diluted in phosphate-buffered saline(PBS) (20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume 2%sodium dodecyl sulfate (SDS) (in H₂O) added (final concentration of 0.2%SDS). An incubation for 6 h at 37° C. resulted in the 16/20-kDa Aβ(1-42)globulomer (short form for globular oligomer) intermediate. The38/48-kDa Aβ(1-42) globulomer was generated by a further dilution withthree volumes of H₂O and incubation for 18 h at 37° C. Cross-linking ofthe 38/48-kDa Aβ(1-42) globulomer was now performed by incubation with 1mM glutaraldehyde for 2 h at 21° C. room temperature (RT) followed byethanolamine (5 mM) treatment for 30 min at RT.

c) Aβ(20-42) Globulomer

1.59 ml of Aβ(1-42) globulomer preparation prepared according to example1a were admixed with 38 ml of buffer (50 mM MES/NaOH, pH 7.4) and 200 μlof a 1 mg/ml thermolysin solution (Roche) in water. The reaction mixturewas stirred at RT for 20 h. Then 80 μl of a 100 mM EDTA solution, pH7.4, in water were added and the mixture was furthermore adjusted to anSDS content of 0.01% with 400 μl of a 1% strength SDS solution. Thereaction mixture was concentrated to approx. 1 ml via a 15 ml 30 kDaCentriprep tube. The concentrate was admixed with 9 ml of buffer (50 mMMES/NaOH, 0.02% SDS, pH 7.4) and again concentrated to 1 ml. Theconcentrate was dialyzed at 6° C. against 1 I of buffer (5 mM sodiumphosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate wasadjusted to an SDS content of 0.1% with a 2% strength SDS solution inwater. The sample was centrifuged at 10000 g for 10 min and theAβ(20-42) globulomer supernatant was withdrawn

d) Aβ(12-42) Globulomer

2 ml of an Aβ(1-42) globulomer preparation prepared according to example1a were admixed with 38 ml buffer (5 mM sodium phosphate, 35 mM sodiumchloride, pH 7.4) and 150 μl of a 1 mg/ml GluC endoproteinase (Roche) Inwater. The reaction mixture was stirred for 6 h at RT, and a further 150μl of a 1 mg/ml GluC endoproteinase (Roche) in water were subsequentlyadded. The reaction mixture was stirred at RT for another 16 h, followedby addition of 8 μl of a 5 M DIFP solution. The reaction mixture wasconcentrated to approx. 1 ml via a 15 ml 30 kDa Centriprep tube. Theconcentrate was admixed with 9 ml of buffer (5 mM sodium phosphate, 35mM sodium chloride, pH 7.4) and again concentrated to 1 ml. Theconcentrate was dialyzed at 6° C. against 1 l of buffer (5 mM sodiumphosphate, 35 mM NaCl) In a dialysis tube for 16 h. The dialysate wasadjusted to an SDS content of 0.1% with a 1% strength SDS solution inwater. The sample was centrifuged at 10000 g for 10 min and theAβ(12-42) globulomer supernatant was withdrawn.

Example 2 Size-Exclusion Chromatography of Different Aβ(1-42) Monomerand Aβ(1-40) Monomer Preparations

Aβ(1-42), 0.1% NH₄OH:

1 mg of Aβ(1-42) (Bachem, catalogue no. H-1368) were dissolved in 500 μlof 0.1% NH₄OH in H₂O and agitated for 1 min at ambient temperature. Thesample was centrifuged for 5 min at 10,000 g. The supernatant wascollected. Aβ(1-42) concentration in the supernatant was determinedaccording to Bradford's method (BIO-RAD).

5 min sample:

20 μl of Aβ(1-42) in the 0.1% NH₄OH containing supernatant were dilutedwith 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 to an Aβ(1-42) concentration of0.2 mg/ml. The sample was incubated for 5 min at ambient temperature.Then 100 μl were analyzed by size exclusion chromatography (SEC).

1 hour sample:

20 μl of Aβ(1-42) in the 0.1% NH₄OH containing supernatant were dilutedwith 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 to an Aβ(1-42) concentration of0.2 mg/ml. The sample was incubated for 1 hour at ambient temperature.Then 100 μl were analyzed by size exclusion chromatography (SEC).

Aβ(1-42), 70% HCOOH:

1 mg of Aβ(1-42) were dissolved in 50 μl 70% HCOOH in H₂O and agitated 1min at ambient temperature. The sample was centrifuged for 5 min at10,000 g. The supernatant was collected. Aβ(1-42) concentration in thesupernatant is determined according to Bradford's method (BIO-RAD).

5 min sample:

2 μl of Aβ(1-42) in 70% HCOOH were diluted to a concentration of 0.2mg/ml Aβ(1-42) with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 and adjusted topH 7.4 with 1 M NaOH. The sample was incubated for 5 min at ambienttemperature. Then 100 μl were analyzed by size exclusion chromatography.

1 hour sample:

2 μl of Aβ(1-42) in 70% HCOOH were diluted to a concentration of 0.2mg/ml Aβ(1-42) with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 and adjusted topH 7.4 with 1 M NaOH. The sample was incubated for 1 hour at ambienttemperature. Then 100 μl were analyzed by size exclusion chromatography.

Aβ(1-42), 0.1% NaOH:

1 mg of Aβ(1-42) (Bachem, catalogue no. H-1368) were dissolved in 500 μlof 0.1% NaOH in H₂O and agitated 1 min at ambient temperature. Thesample was centrifuged for 5 min at 10,000 g. The supernatant wascollected. Aβ(1-42) concentration in the supernatant is determinedaccording to Bradford's method (BIO-RAD).

5 min sample:

20 μl of Aβ(1-42) in 0.1% NaOH were diluted to a concentration of 0.2mg/ml Aβ(1-42) with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4. The sample wasincubated for 5 min at ambient temperature. Then 100 μl were analyzed bysize exclusion chromatography.

1 hour sample:

20 μl of Aβ(1-42) in 0.1% NaOH were diluted to a concentration of 0.2mg/ml Aβ(1-42) with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4. The sample wasincubated for 1 hour at ambient temperature. Then 100 μl were analyzedby size exclusion chromatography.

Aβ(1-40), 0.1% NaOH:

1 mg of Aβ(1-40) (Bachem, catalogue no. H-1194) were dissolved in 500 μlof 0.1% NaOH in H₂O and agitated 1 min at ambient temperature. Thesample is was centrifuged for 5 min at 10,000 g. The supernatant wascollected. Aβ(1-42) concentration in the supernatant was determinedaccording to Bradford's method (BIO-RAD).

5 min sample:

20 μl of Aβ(1-40) in 0.1% NaOH were diluted to a concentration of 0.2mg/ml Aβ(1-40) with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4. The sample wasincubated for 5 min at ambient temperature. Then 100 μl were analyzed bysize exclusion chromatography.

1 hour sample:

20 μl of Aβ(1-40) in 0.1% NaOH were diluted to a concentration of 0.2mg/ml Aβ(1-40) with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4. The sample wasincubated for 1 hour at ambient temperature. Then 100 μl were analyzedby size exclusion chromatography.

Conditions for size exclusion chromatography (SEC):

SEC column: Superose 12 HR 10/300 GL (Amersham, catalogue no.17-5173-01)

Flow: 0.5 ml/min

Paper feed: 0.2 cm/min

Extinction at 214 nm: 0-0.2 absorption units

Mobile phase: 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 Results are shown inFIG. 1.

The preparation of a purely monomeric Aβ-solution is a great challengedue to the strong tendency of the Aβ peptide, especially the Aβ(1-42)monomer, to aggregate into fibrils. Nevertheless, for the screening andcharacterization of anti-Aβ(20-42) globulomers that discriminateAβ(1-42)-monomers and Aβ(1-40)-monomers the best technically achievableAβ-monomeric preparation should be used. Here the effect of the initialsolvent of the Aβ peptide on the aggregation effect after furtherdilution into 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 was tested. The Aβpeptide supplier (Bachem) states in their technical information that theAβ(1-42) should be solubilized in 0.1% NH₄OH. Five minutes at roomtemperature (RT) after solubilizing the Aβ(1-42) in NH₄OH and immediatefurther 1:10 dilution in 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4 asize-exclusion chromatography shows first signs of Aβ(1-42) aggregationto fibrillary precursors with a minor peak at 74kD. Monomeric Aβ(1-42)runs at a major peak with 11 kD and a shoulder at 6kD. After incubationfor one hour at room temperature (RT) the Aβ(1-42) peptide in NH₄OH hasalready aggregated to a high extent to Aβ(1-42) fibrils leading to aloss of detectable material that did not enter the size-exclusionchromatographic column. If 70% formic acid is used as the initialsolvent for Aβ(1-42) peptide a high extent of aggregation after 1 h atRT occurs with only a minor fraction Aβ(1-42) monomer left (note thatthe formic acid itself leads to a high background absorption at theprotein detection wavelength). The best initial solvent for Aβ(1-42) toprevent aggregation is 0.1% NaOH which even after 1 h incubation ofsolubilization and further dilution shows only a minor fraction ofaggregated Aβ(1-42) with the majority of Aβ(1-42) being still monomeric.Aβ(1-40) solubilized initially in 0.1% NaOH shows no signs at all ofaggregation even after 1 h at RT incubation.

Example 3 Semi-Quantitative Analysis Visualized by SDS-PAGE of theDiscrimination of Aβ(20-42) Globulomer Selective Antibodies for Aβ(1-42)Fibrils

Aβ(1-42) fibril preparation:

1 mg of Aβ(1-42) (Bachem, Cat. no.: H-1368) were dissolved in 500 μl0.1% NH₄OH in H₂O and agitated for 1 min at ambient temperature. Thesample was centrifuged for 5 min at 10,000 g. The supernatant wascollected. Aβ(1-42) concentration in the supernatant was determinedaccording to Bradford's method (BIO-RAD).

100 μl of Aβ(1-42) in 0.1% NH₄OH were mixed with 300 μl of 20 mMNaH₂PO₄, 140 mM NaCl, pH 7.4 and adjusted to pH 7.4 with 2% HCl. Thesample was then incubated at 37° C. for 20 hours. Following which thesample was centrifuged for 10 min at 10,000 g. The supernatant wasdiscarded, and the residue was mixed with 400 μl of 20 mM NaH₂PO₄, 140mM NaCl, pH 7.4, resuspended by vigorous agitation (“vortexing”) for 1min and centrifuged for 10 min at 10,000 g. The supernatant wasdiscarded and the residue was mixed with 400 μl of 20 mM NaH₂PO₄, 140 mMNaCl, pH 7.4, resuspended by vigorous agitation (“vortexing”) for 1 minand centrifuged for 10 min at 10,000 g once more. The supernatant wasdiscarded. The residue was resuspended in 380 μl of 20 mM NaH₂PO₄, 140mM NaCl, pH 7.4 and prompted by vigorous agitation (“vortexing”).

Binding of anti-Aβ antibodies to Aβ(1-42) fibrils:

40 μl of Aβ(1-42) fibril preparation were diluted with 160 μl of 20 mMNaH₂PO₄, 140 mM NaCl, 0.05% Tween 20, pH 7.4 and agitated 5 min atambient temperature, then the sample was centrifuged for 10 min at10,000 g. The supernatant was discarded, and the residue was resuspendedin 95 μl of 20 mM NaH₂PO₄, 140 mM NaCl, 0.05% Tween 20, pH 7.4.Resuspension was prompted by vigorous agitation (“vortexing”).

Aliquots of 10 μl of the fibril preparation were each mixed with:

-   -   a) 10 μl 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4    -   b) 10 μl 0.5 μg/μl of 5F7 in 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4    -   c) 10 μl 0.5 μg/μl of 6E10 (Signet Nr.: 9320) in 20 mM NaH₂PO₄,        140 mM NaCl, pH 7.4

The samples were incubated at 37° C. for 20 hours, then centrifuged for10 min at 10,000 g. The supernatants were collected and mixed with 20 μlof SDS-PAGE sample buffer. The residues were mixed with 50 μl of 20 mMNaH₂PO₄, 140 mM NaCl, 0.025% Tween 20, pH 7.4 and resuspended by“vortexing”, then the samples were centrifuged for 10 min at 10,000 g.The supernatants were discarded, and the residues were mixed with 20 μl20 mM NaH₂PO₄, 140 mM NaCl, 0.025% Tween 20, pH 7.4, then with 20 μl ofSDS-PAGE sample buffer. The samples were applied to a 4-20% Tris/glycinegel for electrophoresis.

Parameters for SDS-PAGE:

-   -   SDS sample buffer. 0.3 g SDS        -   4 ml 1 M Tris/HCl pH 6.8        -   8 ml glycerine        -   1 ml 1% bromphenol blue in ethanol        -   Fill with H₂O ad 50 ml    -   4-20% Tris/Glycine Gel: (Invitrogen, Cat. no.: EC6025BOX)    -   Electrophoresis buffer: 7.5 g Tris        -   36 g Glycine        -   2.5 g SDS        -   Fill with H₂O ad 2.5 l    -   The gel is run at a constant current of 20 mA.    -   Staining of the gels: Coomassie Blue R250

Results are shown in FIG. 2.

Semiquantitative analysis of different anti-Aβ antibodies and theirdiscrimination of Aβ(1-42) fibrils. Positions of antibodies, Aβ(1-42)fibrils and Aβ(1-42) monomers are marked at the edge of the gel. Due totheir size, Aβ(1-42) fibrils cannot enter the SDS-PAGE gel and can beseen in the gel slot.

-   -   1. Marker    -   2. Aβ(1-42) fibril preparation; control    -   3. Aβ(1-42) fibril preparation; +mAb 5F7; 20 h 37° C.;        supernatant    -   4. Aβ(1-42) fibril preparation; +mAb 5F7; 20 h 37° C.; pellet    -   5. Aβ(1-42) fibril preparation; +mAb 6E10; 20 h 37° C.;        supernatant    -   6. Aβ(1-42) fibril preparation; +mAb 6E10; 20 h 37° C.; pellet

The relative binding to fibril type Aβ was evaluated from SDS-PAGEanalysis by measuring the Optical Density (OD) values from the HeavyChain of the antibodies in the fibril bound (pellet-fraction) and thesupernatant fractions after centrifugation. Antibodies that have boundto the As fibrils should be co-pelleted with the Aβ-fibrils andtherefore are found in the pellet fraction whereas non-Aβ-fibril bound(free) antibodies are found in the supernatant. The percentage ofantibody bound to Aβ-fibrils was calculated according to the followingformula:

Percent antibody bound toAβ-fibrils=OD_(fibril fraction)×100%/(OD_(fibril fraction)+OD_(supernatant fraction)).

This procedure was performed for the mAbs 6E10 (Signet, Cat. no.: 9320),5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1.

In the Alzheimer disease brain the Aβ fibrils are a major component ofthe total Aβ peptide pool. By attacking these fibrils by antiAβ-antibodies the risk of negative side effects is elevated due to aliberation of high amounts of Aβ which subsequently may increase therisk of microhaemorrhages. An increased risk for microhaemorrhages wasobserved in an active Immunization approach with fibrillar aggregates ofthe Aβ peptide (Bennett and Holtzman, 2005, Neurology, 64, 10-12;Orgogozo J, Neurology, 2003, 61, 46-54; Schenk et al., 2004, Curr OpinImmunol, 16, 599-606).

In contrast to the commercially available antibody 6E10 (Signet 9320)which recognizes a linear Aβ-epitope between AA1-17, the Aβ(20-42)globulomer selective antibody 5F7 (which actually has the lowestselectivity for Aβ(20-42) globulomers over other Aβ-forms) does not bindto Aβ(1-42) fibrils in an co-pelleting experiment. This is shown by thefact that the 5F7 antibody after an incubation with Aβ(1-42) fibrilsremains after a pelleting step in the supernatant and is not co-pelletedbecause of being bound to the Aβ(1-42) fibrils.

Example 4 Analysis of Cognitive Performance in Mice by Means of anObject Recognition Test after Active Immunization with Aβ(1-42) Monomer(0.1% NH₄OH), Aβ(1-42) Globulomer or Aβ(20-42) Globulomer in Comparisonto Wild Type

In these experiments mice overexpressing human APP with a point mutationwere used. The point mutation refers to amino acid 717 (substitution ofisoleucine for valine) and has been found in a London family where itleads to onset of AD before the beginning of the sixth decade of life(Mullan et al., Nature Genetics 2 (1992) 340-342). The transgenic mice,herein referred to as APP/L, were created by and first described inLeuven (Moechars et al., J. Biol Chem. 274 (1999) 6483-6492). FemaleAPP/L mice were subjected to active immunization at 6 weeks of age.

The mice received either 100 μg of Aβ(1-42) monomer (0.1% NH₄OH),Aβ(1-42) globulomer or Aβ(20-42) globulomer in phosphate-buffered saline(PBS) mixed with an equal amount of complete Freund's adjuvantintraperitoneally, followed by booster injections with the same a mountof antigene in incomplete Freund's adjuvant every third week for threemonths. Throughout the time course of the experiment the animals werekept under standard conditions in a reverted day/night cycle (14 hoursof light beginning at 7 pm/10 hours of darkness). Body weight gain overthe time of the experiment was as expected and did not differ from acontrol group which received PBS/adjuvant alone, suggesting that theantigen treatments were well tolerated.

At 4.5 month of age cognitive ability of the mice was tested by anobject recognition test as described in the art (Dewachter et al.Journal of Neuroscience 22 (2002) 3445-3453). To this end, mice wereaccustomed to an arena and then exposed for 10 minutes to an acquisitionphase during which they were individually placed in the arena which nowcontained two identical elements (blue pyramid, green cube, yellowcylinder of similar size, ca. 4 cm). The duration and frequency withwhich the mouse explored the objects were recorded. During retentionphase, 2.5 h later, mice were returned to the arena which now contained,in addition to the known object, an unknown object randomly selectedfrom the other objects. Recognition of the new object was recorded asthe time during which the mouse was exploring the old object relative tototal time (exploration of old and new object). The “recognition index”expresses this relation (time for new object/total time). A mouse whichdoes not remember the known object will consider it as equallyinteresting as the new object and spend an equal amount of time onexploring it, in other words, will show a recognition index of 50%. Amouse which remembers the known object will consider it as notinteresting and therefore show a significantly higher recognition index.APP/L mice are known to be cognitively deficient at 4.5 months of ageand exhibit a recognition index in the dimension of the random level,i.e. 50%.

Results are shown in FIG. 3.

Object recognition test in mice. The test reports recognition of a knownobject in comparison to an unknown one, measured in terms of explorativebehaviour during a 10 minute test phase. The recognition Index isdefined as the percentage of time which the mouse spends on exploringthe unknown object relative to the time spent on exploring both objects.The known object was explored by the mouse during a 10 minuteacquisition phase three hours before the test phase. Five groups of mice(number n given below the columns) were compared. Normal C57BI/6 mice(wild type) show a high RI significantly different from random level(50%, i.e. equal times of exploration spent on both the known and theunknown object) (**=p<0.001; Student's t-test). The other four groups ofAPP transgenic mice were subjected to active immunisation three monthsbefore. The immunogens used were Aβ(1-42) monomer, Aβ(1-42) globulomerand Aβ(20-42) globulomer. Phosphate-buffered saline (PBS) was used ascontrol. Significant differences between PBS and the other groups areindicated with circles: °=p<0.05; °°=p<0.01 (post-hoc t-test afterp<0.05 in ANOVA).

APP/L mice are known to show, in contrast to non-transgenic mice, acognitive deficiency at 4.5 months of age, scoring results close torandom level (i.e. 50% recognition index). In fact, the PBS-treated miceshowed random behaviour, in contrast to non-transgenic mice (wild type).Immunization with native Aβ(1-42) globulomer as well as with Aβ(20-42)globulomer resulted in significantly Improved object recognition inAPP/L mice.

As both globulomer preparations (native and truncated) resulted inmemory improvement in APP transgenic animals and even superiorrecognition in animals treated with Aβ(20-42) globulomer it isreasonable to assume that induction of antibodies against truncatedAβ(20-42) globulomer will produce the best result, and that passiveimmunisation with antibodies reacting specifically with this speciesrepresents the optimal strategy of treatment.

Example 5 Dot-Blot Analysis of the Antibody Profile for DifferentAβ-Forms after an Active Immunization of APP/L Tg mice with Aβ(20-42)Globulomer

After immunization of mice (compare example 4) of APP/L mice (Moecharset al., 1999, J. Biol. Chem. 274, 6483-6492) with different forms of AP,plasma samples were assessed for anti-Aβ antibodies To this end,dilution series of the individual Aβ(1-42) forms ranging from 100pmol/μl to 0.01 pmol/μl in PBS supplemented with 0.2 mg/ml BSA weremade. 1 μl of each sample was blotted onto a nitrocellulose membrane.For detection the corresponding mouse plasma samples were used (diluted1:400). Immunostaining was done using alkaline phosphatase conjugatedanti-mouse-IgG and the staining reagent NBT/BCIP.

Aβ-standards for dot-blot:

1. Aβ(1-42) Globulomer

The preparation of the Aβ(1-42) globulomer is described in example 1a.

2. HFIP Pretreated Aβ(1-42) Monomer in Pluronic F68

3 mg of Aβ(1-42), (Bachem Inc; cat no. H-1368) were dissolved in 0.5 mlHFIP (6 mg/ml suspension) in an 1.7 ml Eppendorff tube and was shaken(Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37° C. till a clearsolution was obtained. The sample was dried in a SpeedVac concentrator(1.5 h) and resuspended in 13.2 μl DMSO, shaken for 10 sec., followed bysonification (20 sec), and shaking (e g. in Eppendorff Thermo mixer,1400 rpm) for 10 min. 6 ml of 20 mM NaH₂PO₄; 140 mM NaCl; 0.1% PluronicF68; pH 7.4 were added and stirred for 1 h at room temperature. Thesample was centrifuged for 20 min at 3000 g. The supernatant wasdiscarded and the precipitate solved in 0.6 ml 20 mM NaH₂PO₄; 140 mMNaCl; 1% Pluronic F68, pH 7.4. 3.4 ml of H₂O were added and stirred for1 h at room temperature followed by 20 min centrifugation at 3000 g.Eight aliquots of each 0.5 ml of the supernatant were stored at −20° forfurther use.

3. Aβ(20-42) Globulomer

The preparation of the Aβ(20-42) globulomer is described in example 1c.

4. Aβ(12-42) Globulomer

The preparation of the Aβ(12-42) globulomer is described in example 1d.

5. HFIP Pretreated Aβ(1-40) Monomer, 5 mM in DMSO

1 mg Aβ(1-40), (Bachem Inc, cat. no. H-1194) were suspended in 0.25 mlHFIP (4 mg/ml suspension) in an Eppendorff tube. The tube was shaken(e.g. In Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37° C. to get aclear solution and afterwards dried in a speed vac concentrator (1.5 h).The sample was redissolved in 46 μl DMSO (21.7 mg/ml solution=5 mM),shaken for 10 sec and subsequently sonicated for 20 sec. After 10 minshaking (e.g. in Eppendorff Thermo mixer, 1400 rpm) the sample is storedat −20° C. for further use.

6. Aβ(1-42) Monomer, 0.1% NH₄OH

1 mg Aβ(1-42) (Bachem Inc., cat no. H-1368) were dissolved in 0.5 ml0.1% NH₄OH in H₂O (freshly prepared) (=2 mg/ml) and immediately shakenfor 30 sec. at room temperature to get a clear solution. The sample wasstored at −20° C. for further use.

7. Aβ(1-42) Fibrils

1 mg Aβ(1-42) (Bachem Inc. Catalog Nr.: H-1368) were dissolved in 500 μlaqueous 0.1% NH₄OH (Eppendorff tube) and the sample was stirred for 1min at room temperature. 100 μl of this freshly prepared Aβ(1-42)solution were neutralized with 300 μl 20 mM NaH₂PO₄; 140 mM NaCl, pH7.4. The pH was adjusted to pH 7.4 with 1% HCl. The sample was incubatedfor 24 h at 37° C. and centrifuged (10 min at 10000 g). The supernatantwas discarded and the fibril pellet resuspended with 400 μl of 20 mMNaH₂PO₄; 140 mM NaCl, pH 7.4 by vortexing for 1 min.

8. sAPPα

Supplied from Sigma (cat.no. 89564; 25 μg in 20 mM NaH₂PO₄; 140 mM NaCl;pH 7.4). The sAPPα was diluted with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4,0.2 mg/ml BSA to 0.1 mg/ml (=1 pmol/μl).

Materials Dot Blot: Aβ-Standards:

-   -   Serial dilution of Aβ-antigens in 20 mM NaH₂PO₄, 140 mM NaCl, pH        7.4+0.2 mg/ml BSA        -   1) 100 pmol/μl        -   2) 10 pmol/μl        -   3) 1 pmol/μl        -   4) 0.1 pmol/μl        -   5) 0.01 pmol/μl

Nitrocellulose:

-   -   Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45        μm); BIO-RAD

Anti-Mouse-AP:

-   -   AQ330A (Chemicon)

Detection Reagent:

-   -   NBT/BCIP Tablets (Roche)

Bovine Serum Albumin, (BSA):

-   -   A-7888 (SIGMA)

Blocking Reagent:

-   -   5% low fat milk in TBS

Buffer Solutions:

-   -   TBS    -   25 mM Tris/HCl-buffer pH 7.5    -   +150 mM NaCl    -   TTBS    -   25 mM Tris/HCl-buffer pH 7.5    -   +150 mM NaCl    -   +0.05% Tween 20    -   PBS+0.2 mg/ml BSA    -   20 mM NaH₂PO₄ buffer pH 7.4    -   +140 mM NaCl    -   +0.2 mg/ml BSA

Antibody Solution I:

-   -   Mouse plasma samples from an active immunization study with        Aβ(20-42) globulomer (1:400 diluted in 20 ml 1% low fat milk in        TBS)

Antibody Solution II:

-   -   1:5000 dilution    -   Anti-Mouse-AP in 1% low fat milk in TBS

Dot-Blot-Procedure:

-   -   1) 1 μl each of the different A-standards (in their 5 serial        dilutions) were dotted onto the nitrocellulose membrane in a        distance of approximately 1 cm from each other.    -   2) The Aβ-standards dots were allowed to dry on the        nitrocellulose membrane on air for at least 10 min at room        temperature (RT) (=dot blot)    -   3) Blocking:        -   The dot blot was incubated with 30 ml 5% low fat milk in TBS            for 1.5 h at RT.    -   4) Washing:        -   The blocking solution was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.    -   5) Antibody solution I:        -   The washing buffer was discarded and the dot blot was            incubated with antibody solution I overnight at RT.    -   6) Washing:        -   The antibody solution I was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TBS for 10 min at RT.    -   7) Antibody solution II:        -   The washing buffer was discarded and the dot blot was            incubated with antibody solution II for 1 h at RT    -   8) Washing:        -   The antibody solution II was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TBS for 10 min at RT.    -   9) Development:        -   The washing solution was discarded. 1 tablet NBT/BCIP was            dissolved in 20 ml H₂O and the dot blot was incubated for 5            min with this solution. The development was stopped by            intensive washing with H₂O.

Results are shown in FIG. 4.

Dot blot analysis of anti-Aβ antibodies produced after activeimmunization of mice with Aβ(20-42) globulomer to assess theirspecificity towards different forms of AP. The individual forms of Aβwere blotted in serial dilutions and incubated with the correspondingmouse plasma containing anti AP-antibodies produced during immunereaction. The individual dot blots correspond to different individualsof Immunized mice.

-   -   1. Aβ(1-42) globulomer    -   2. Aβ(1-42) monomer, HFIP pretreated, in 0.1% Pluronic F68    -   3. Aβ(20-42) globulomer    -   4. Aβ(12-42) globulomer    -   5. Aβ(1-40) monomer, HFIP pretreated, 5 mM in DMSO    -   6. Aβ(1-42) monomer, 0.1% NH₄OH    -   7. Aβ(1-42) fibril preparation    -   8. sAPPα (Sigma); (first dot 1 pmol)

In the active immunization study of APP/L Tg-mice it was shown thatimmunization with Aβ(20-42) globulomer leads to the best result inalleviating a cognitive impairment in these mice compared to PBStreatment Plasma samples from APP/L Tg mice after being activelyimmunized with Aβ(20-42) globulomer exhibit an antibody profile(predominant recognition of Aβ(20-42) globulomer and Aβ(12-42)globulomer) which resembles that of the Aβ(20-42) globulomer mAbsclaimed herein.

Example 6 Concentration of Soluble and Insoluble Aβ(1-42) and Aβ(1-40)Peptide in Brain ExTracts of Actively Immunized APP/PS1 Tg Mice withEither Aβ(1-42) Monomer, Aβ(1-42) Globulomer, Aβ(20-42) Globulomer orVehicle as Control

40 female mice of a double transgenic mouse model of Alzheimer's Disease(APP/PS1 Tg mice) in FVBxC57BI/6J background with an age of 4 monthswere used for this study. The APP/PS1 Tg mice contain the 695 amino acidform of human APP with the V7171 mutation (position referring to thelongest APP-isoform) and in addition the human Presenilin 1 gene withthe A264E mutation. Both genes are under control of the Thyl promotor.The mice were generated and characterized in one of the foundinglaboratories of reMYND, the Experimental Genetics Group, CampusGasthuisberg, Catholic University Leuven, by Prof. Fred Van Leuven etal.

All mice were genotyped by polymerase chain reaction (PCR) at the age of3 weeks and received a unique identity number, once the PCR results wereknown.

Mice had free access to pre-filtered and sterile water (UV-lamp) andstandard mouse chow.

The food was stored under dry and cool conditions in a well-ventilatedstorage room. The amount of water and food was checked daily, suppliedwhen necessary and by default refreshed twice a week.

Mice were housed under a reversed day-night rhythm: 14 hours light/10hours darkness starting at 7 p.m., in standard metal cages type RVS T2(area of 540 cm²). The cages are equipped with solid floors and layer ofbedding litter. The number of mice per cage was limited in accordancewith legislation on animal welfare. Five days before the onset of thebehaviour test, mice were re-caged in macrolon Type 2 cages andtransported to the laboratory in order to adapt to the laboratoryenvironment in preparation to the behaviour test.

The mice received either 100 μg of Aβ(1-42) monomer (0.1% NH₄OH),Aβ(1-42) globulomer or Aβ(20-42) globulomer in phosphate-buffered saline(PBS) mixed with an equal amount of complete Freund's adjuvantintraperitoneally, followed by booster injections with the same amountof antigene in incomplete Freund's adjuvant every third week for fourmonths.

Biochemistry

The Aβ(1-40) and Aβ(1-42) in the soluble fraction of 1 hemisphere wasdetermined by ELISA. In addition the Aβ(1-40) and Aβ(1-42) of theinsoluble membrane fraction of 1 hemisphere was determined by ELISA

The mice were anaesthetized with a 2:1:1 mixture of Ketalar (ketamin),Rompun (xylazin 2%) and atropin and flushed trans-cardially withphysiological serum at 4° C. This was performed to remove blood from thebrain vessels, a procedure which has no influence on organ integrity.

Cerebrospinal fluid (CSF) was collected via an incision in the neckmuscles, between the skull and the first cervical vertebrae. A punctureinto the cistema magna was given with a 26 gauge needle and 10-20 μl ofCSF was collected with a fine glass pipette.

Blood was collected via a heart puncture and a 1 ml syringe intoheparin-coated Eppendorf tubes. The blood was centrifuged at 14, 000 rpmat 4° C. for 5 minutes. The serum was stored at −70° C.

The mice were flushed transcardially with physiological serum at 4° C.

The brain was removed from the cranium and hindbrain and forebrain wereseparated by a cut in the coronal/frontal plane. The cerebellum wasdischarged. The forebrain was divided evenly into left and righthemisphere by using a midline sagittal cut

One hemisphere was immediately immersed in liquid nitrogen and stored at−70° C. until biochemical analysis.

Homogenization and Fractionation of One Brain Hemisphere

Brains were homogenized using a Potter, a glass tube (detergent free, 2cm³) and a mechanical homogenizer (650 rpm). A volume of 6.5×½ brainweight of freshly prepared 20 mM Tris/HCl buffer (pH 8.5) withProteinase Inhibitors (1 tablet per 50 ml Tris/HCl buffer, Complete™,Roche, Mannheim, Germany) was used as homogenization buffer.

Samples were transferred from −70° C. into a sample holder with liquidnitrogen and each individual sample was pre-warmed by incubation on thebench for a few seconds prior to homogenization. The homogenates werecollected in Beckman centrifuge tubes TLX and collected on ice prior tocentrifugation. Between two samples, the Potter and the glass tube wererinsed carefully with distilled water (AD) without detergents and driedwith absorption paper.

Samples were centrifuged in a pre-cooled ultracentrifuge (Beckman,Mannheim, Germany) for 1 hour and 20 minutes at 48000 rpm (135.000×g) at4° C. Due to a limited number of centrifuge holders (N=8), samples weresorted by brain weight (to equilibrate the centrifuge) and randomized inorder to divide the different treatment groups over the differentcentrifugation sessions.

The supernatant (soluble fraction containing secreted APP and amyloidpeptides) was separated from the pellet (membrane fraction containingmembrane-bound APP-fragments and plaque-associated amyloid peptides incase of aged mice). The supernatant was divided over two tubes of whichone was stored at −20° C. as back-up and the other was processed furtherfor column chromatography to concentrate the amyloid peptides.

Brain weights, volume Tris/HCl buffer used, centrifugation sessions(marked by colour) and volume soluble fraction used for columnchromatography are given exemplary in the following table.

Weight V Tris 50% Sample brain (W) (=W × 6.5) V Tris N^(o) TreatmentMouse ID N^(o) (mg) (μl) (μl) 19 X TAB.TPF 1305 157.8 1026 513 21 XTAB.TPF 1335 160.2 1041 521

Small reversed phase columns (C18-Sep-Pack Vac 3cc cartridges, Waters,Mass., MA) were mounted on a vacuum system and washed with 80%acetonitrile in 0.1% trifluoroacetic acid (A-TFA) followed with 0.1% TFAtwice. Then the samples were applied and the columns were washedsuccessively with 5% and 25% A-TFA. Amyloid peptides were eluted with75% A-TFA and the eluates were collected in 2 ml tubes on ice. Eluateswere freeze-dried in a SpeedVac concentrator (Savant, Farmingdale, N.Y.)overnight and resolved in 330 μl of the sample diluent furnished withthe ELISA kits.

The pellets were further fractionated into different membrane fractions:membrane fraction A (MFA), membrane fraction B (MFB) containing fulllength APP and membrane fraction C (MFC) containing plaque associatedamyloid. Therefore the pellets were dissolved in TBS buffer withproteinase inhibitors (1 tablet per 50 ml TBS buffer, Complete™, Roche,Mannheim, Germany) and the MFA was divided over two tubes of which onewas stored at −20° C. as backup. 60% of MFA was further processed withaddition of NP40 (2% of final volume) and Triton X-100 (2% of finalvolume) in TBS with proteinase inhibitors and centrifuged for one hourat 27.000 rpm (98,000×g) in a Beckman ultracentrifuge at 4° C. using aswing-out rotor (SW60). The supernatant (MFB) was separated from thepellet (MFC) and both were stored at −20° C.

Brain weights, 60% of the brain weight, the volumes TBS+PI+NP40+TritonX-100 buffer used and the centrifugation sessions (marked by colour) aregiven exemplary in the following table.

⅗ × Volume Weight Weight buffer = Sample brain (W) brain ⅗ W × 15 N^(o)Treatment Mouse ID N^(o) (mg) (mg) (μl) 19 X TAB.TPF 1305 157.8 95 142021 X TAB.TPF 1335 160.2 96 1442

ELISA of Human Aβ in the Soluble Fraction of One Hemisphere

To quantify the amount of human Aβ(1-40) and human Aβ(1-42) in thesoluble fraction of the brain homogenates and/or in cerebrospinal fluid(CSF), commercially available Enzyme-Linked-Immunosorbent-Assay (ELISA)kits were used (h Amyloid β40 or β42 ELISA high sensitive, The GeneticsCompany, Zurich, Switzerland). The ELISA was performed according to themanufacturer's protocol. Briefly, standards (a dilution of syntheticAβ(1-40) or Aβ(1-42)) and samples were prepared in a 96-wellpolypropylene plate without protein binding capacity (Greiner bio-one,Frickenhausen, Germany). The standard dilutions with finalconcentrations of 1000, 500, 250, 125, 62.5, 31.3 and 15.6 pg/ml and thesamples were prepared in the sample diluent, furnished with the ELISAkit, to a final volume of 60 μl. Since amyloid levels increase with theage of the mouse and since the actual evaluation requires that thereadings of the samples are within the linear part of the standardcurve, the samples for Aβ(1-40) analysis were diluted 1:3, the samplesfor Aβ(1-42) analysis were diluted 1:6.

Samples, standards and blanks (50 μl) were added to the anti-Aβ-coatedpolystyrol plate (capture antibody selectively recognizes the C-terminalend of the antigen) in addition with a selective anti-An-antibodyconjugate (biotinylated detection antibody) and incubated overnight at4° C. in order to allow formation of theantibody-Amyloid-antibody-complex. The following day, aStreptavidine-Peroxidase-Conjugate was added, followed 30 minutes laterby the addition of a TMB/peroxide mixture, resulting in the conversionof the substrate into a coloured product. This reaction was stopped bythe addition of sulfuric acid (1 M) and the colour intensity wasmeasured by means of photometry with an ELISA-reader with a 450 nmfilter. Quantification of the Aβ content of the samples was obtained bycomparing absorbance to the standard curve made with synthetic Aβ(1-40)or Aβ(1-42).

ELISA human Aβ in the insoluble fraction of one hemisphere To quantifythe amount of human Aβ(1-40) and human Aβ(1-42) in the insolublemembrane fraction of the brain homogenates, the MFC samples were furtherprocessed and dissolved in 8M Guanidine in 80 mM Tris/HCl. Subsequentlysamples were incubated for 3 hours in a thermomixer at 25° C. andpipetted up and down with a 100 μl pipette every hour to dissolve theMFC pellet into the guanidine buffer Finally samples were centrifugedfor only 1 minute at 4000 rpm to remove debris.

Brain weight, the weight of the MFC pellet and the volume of 8Mguanidine buffer are given exemplary in the following table.

Weight Weight pellet of Volume brain MFC 8M guanidine Sample Mouse (W)(WMFC) (WMFC × 1.6) N^(o) Treatment ID N^(o) (mg) (40% brain) (μl) 19 XTAB.TPF 157.8 63 101 1305 2 X TAB.TPF 160.2 64 103 1335

To quantify the amount of human Aβ(1-40) and human Aβ(1-42) in the finalsamples, commercially available Enzyme-Unked-lmmunosorbent-Assay (ELISA)kits were used (h Amyloid 40 or β42 ELISA high sensitive, The GeneticsCompany, Zurich, Switzerland). The ELISA was performed according to themanufacturer's protocol, except for the preparation of the standards (adilution of synthetic Aβ(1-40) or Aβ(1-42)). The samples were preparedin the sample diluent, furnished with the ELISA kit, to a final volumeof 60 μl. Since guanidine influences the OD-values of the standardcurve, the standard dilutions with final concentrations of 1000, 500,250, 125, 62.5, 31.3 and 15.6 pg/ml were prepared in sample diluent withthe same concentration guanidine as for the samples. This was performedin a 96-well polypropylene plate without protein binding capacity(Greiner bio-one, Frickenhausen, Germany).

Since amyloid levels increase with the age of the mouse and since theactual evaluation requires that the readings of the samples are withinthe linear part of the standard curve, the samples for insolubleAβ(1-40) and insoluble Aβ(1-42) analysis were diluted 1:500.

Samples, standards and blanks (50 μl) were added to the anti-Aβ-coatedpolystyrol plate (capture antibody selectively recognizes the C-terminalend of the antigen) in addition with a selective anti-Aβ-antibodyconjugate (biotinylated detection antibody) and incubated overnight at4° C. in order to allow formation of theantibody-Amyloid-antibody-complex. The following day, astreptavidin-peroxidase conjugate was added, followed 30 minutes laterby the addition of a TMB/peroxide mixture, resulting in the conversionof the substrate into a coloured product. This reaction was stopped bythe addition of sulfuric acid (1M) and the colour intensity was measuredby means of photometry with an ELISA-reader with a 450 nm filter.Quantification of the Aβ content of the samples was obtained bycomparing absorbance to the standard curve made with synthetic Aβ(1-40)or Aβ(1-42)

Results are shown in FIG. 5

Concentration of soluble and insoluble Aβ(1-42) and Aβ(1-40) peptide inbrain extracts of actively immunized APP/PS1 Tg-mice with eitherAβ(1-42) monomer (0.1% NH₄OH), Aβ(1-42) globulomer, Aβ(20-42) globulomeror vehicle as control.

In the soluble and insoluble fraction of a brain extract of APP/PS1Tg-mice actively Immunized with Aβ(20-42) globulomer the level ofAβ(1-40)- and Aβ(1-42)-peptide is not significantly different to thevehicle control. In contrast, immunization with Aβ(1-42) globulomer andAβ(1-42) monomer leads to a reduction in brain Aβ(1-40)- andAβ(1-42)-levels. This shows that an Aβ(20-42) globulomer directedimmunization approach does not alter the total Aβ-brain levelssignificantly but nonetheless is effective in alleviating the Aβ-peptiderelated cognitive impairments (see example 4).

Example 7 Analysis of Cognitive Performance by Object Recognition Testin APP/L Transgenic Mice after Passive Immunization with Anti-Aβ(20-42)Globulomer Antibodies

In these experiments mice overexpressing human APP with a point mutationwere used. The point mutation refers to amino acid 717 (substitution ofisoleucine for valine) and has been found in a London family where itleads to onset of AD before the beginning of the sixth decade of life(Mullan et al., Nature Genetics 2 (1992) 340-342). The transgenic mice,herein referred to as APP/L, were created by and first described inLeuven (Moechars et al., J. Biol. Chem. 274 (1999) 6483-6492). FemaleAPP/L mice were subjected to passive immunization at 3 months of age.Mice received 250 μg of any of the monoclonal mouse antibodies 5F7,10F11 or 7C6 in 100 μl of phosphate-buffered saline (PBS). Throughoutthe time course of the experiment the animals were kept under standardconditions in a reverted day/night cycle (14 hours of light beginning at7 pm/10 hours of darkness). They tolerated passive immunization well,without any signs of adverse effects.

After the third injection (day 15 of experiment) cognitive ability ofthe mice was tested by an object recognition test as described in theart (Dewachter et al. Journal of Neuroscience 22 (2002) 3445-3453). Tothis end, mice were accustomed to an arena and then exposed for 10minutes to an acquisition phase during which they were individuallyplaced in the arena which now contained two identical elements (greencube or orange cylinder of similar size, ca. 4 cm). The duration andfrequency with which the mouse explored the objects were recorded.During retention phase, 2.5 h later, mice were returned to the arenawhich now contained, in addition to the known object, the other object.Recognition of the new object was recorded as the time during which themouse was exploring the old object relative to total time (explorationof old and new object). The “recognition index” expresses this relation(time for new object/total time). A mouse which does not remember theknown object will consider it as equally interesting as the new objectand spend an equal amount of time on exploring it, in other words, willshow a recognition index of 50%. A mouse which remembers the knownobject will consider it as not interesting and therefore show asignificantly higher recognition index. APP/L mice are known to becognitively deficient at 4.5 months of age and exhibit a recognitionindex in the dimension of the random level, i.e. 50%.

Results are shown in FIG. 6.

Object recognition test in mice. The test reports recognition of a knownobject in comparison to an unknown one, measured in terms of explorativebehaviour during a 10 minute test phase. The recognition index isdefined as the percentage of time which the mouse spends on exploringthe unknown object relative to the time spent on exploring both objects.The known object was explored by the mouse during a 10 minuteacquisition phase 2.5 hours before the test phase.

a) APP transgenic mice were immunized once a week for three weeks byintraperitoneal injection of 250 μg of the antibody 5F7 (n=9), theantibody 10F11 (n=11) or the antibody 7C6 (n=11); control animalsreceived PBS (n=6). Significant differences from random level (50%, i.e.equal time of exploration spent on the known and the unknown object) areindicated with asterisks. *=p<0.05 (t-test)

b) Comparison of all mice treated with antibodies (5F7, 10F11 and 7C6;(n=31)) and mice treated with phosphate-buffered saline (PBS; n=6). TheRI of the antibody-treated group different significantly from randomlevel (**=P<0.01; t-Test).

APP/L mice are known to be cognitively deficient at 4.5 months of ageand exhibit a recognition index in the dimension of the random level,i.e. 50%.

Indeed the PBS-treated mice showed random behaviour. Passiveimmunization with all three antibodies (5F7, 10F11 and 7C6) resulted ina markedly increased recognition index. When compared as a pooled groupagainst the controls, the recognition index is significantly increased.This beneficial effect on memory performance of APP/L mice afteradministration of all three antibodies suggests that an antibody againsttruncated Aβ(20-42) globulomer is sufficient to achieve cognitiveimprovement.

Example 8 Dot-Blot Profile of the Selectivity of the Anti-Aβ(20-42)Globulomer Antibodies

In order to characterize the selectivity of the monoclonal antiAβ(20-42) globulomer antibodies they were probed for recognition withdifferent Aβ-forms. To this end, serial dilutions of the individualAβ((1-42) forms ranging from 100 pmol/μl to 0.01 pmol/μl in PBSsupplemented with 0.2 mg/ml BSA were made. 1 μl of each sample wasblotted onto a nitrocellulose membrane. For detection the correspondingantibody was used (0.2 μg/ml). Immunostaining was done using peroxidaseconjugated anti-mouse-IgG and the staining reagent BM Blue POD Substrate(Roche).

Aβ-Standards for Dot-Blot: 1. Aβ(1-42) Monomer, 0.1% NH₄OH

1 mg Aβ(1-42) (Bachem Inc., cat. no. H-1368) were dissolved in 0.5 ml0.1% NH₄OH In H₂O (freshly prepared) (=2 mg/ml) and immediately shakenfor 30 sec at room temperature to get a clear solution. The sample wasstored at −20° C. for further use.

2. Aβ(1-40) Monomer, 0.1% NH₄OH

1 mg Aβ(1-40) (Bachem Inc., cat. no. H-1368) were dissolved in 0.5 ml0.1% NH₄OH In H₂O (freshly prepared) (=2 mg/ml) and immediately shakenfor 30 sec. at room temperature to get a clear solution. The sample wasstored at −20C for further use.

3. Aβ(1-42) Monomer, 0.1% NaOH

2.5 mg Aβ(1-42) (Bachem Inc., cat. no. H-1368) were dissolved in 0.5 ml0.1% NaOH in H₂O (freshly prepared) (=5 mg/ml) and immediately shakenfor 30 sec. at room temperature to obtain a clear solution. The samplewas stored at −20° C. for further use.

4. Aβ(1-40) Monomer, 0.1% NaOH

2.5 mg Aβ(1-40) (Bachem Inc, cat. no. H-1368) were dissolved in 0.5 ml0.1% NaOH in H₂O (freshly prepared) (=5 mg/ml) and Immediately shakenfor 30 sec. at room temperature to obtain a clear solution. The samplewas stored at −20° C. for further use.

5. Aβ(1-42) Globulomer

The preparation of the Aβ(1-42) globulomer is described in example 1a.

6. Aβ(12-42) Globulomer

The preparation of the Aβ(12-42) globulomer is described in example 1d.

7. Aβ(20-42) Globulomer

The preparation of the Aβ(20-42) globulomer is described in example 1c.

8. Aβ(1-42) Fibrils

1 mg Aβ(1-42) (Bachem Inc. cat. no.: H-1368) were solved in 500 μlaqueous 0.1% NH₄OH (Eppendorff tube) and the sample was stirred for 1min at room temperature. 100 μl of this freshly prepared Aβ(1-42)solution were neutralized with 300 μl 20 mM NaH₂PO₄; 140 mM NaCl, pH7.4. The pH was adjusted to pH 7.4 with 1% HCl. The sample was incubatedfor 24 h at 37° C. and centrifuged (10 min at 10000 g) The supernatantwas discarded and the fibril pellet resuspended with 400 μl 20 mMNaH₂PO₄; 140 mM NaCl, pH 7.4 by vortexing for 1 min.

9. sAPPα

Supplied by Sigma (cat.no. S9564; 25 μg in 20 mM NaH₂PO₄; 140 mM NaCl;pH 7.4). The sAPPα was diluted to 0.1 mg/ml (=1 pmol/μl) with 20 mMNaH₂PO₄, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA.

Materials for Dot Blot: Aβ-Standards:

-   -   Serial dilution of Aβ antigens in 20 mM NaH₂PO₄, 140 mM NaCl, pH        7.4+0.2 mg/ml BSA        -   1) 100 pmol/μl        -   2) 10 pmol/μl        -   3) 1 pmol/μl        -   4) 0.1 pmol/μl        -   5) 0.01 pmol/μl

Nitrocellulose:

-   -   Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45        μm); BIO-RAD

Anti-Mouse-POD:

-   -   Cat no: 715-035-150 (Jackson Immuno Research)

Detection Reagent:

-   -   BM Blue POD Substrate, precipitating (Roche)

Bovine Serum Albumin, (BSA):

-   -   Cat no: A-7888 (SIGMA)

Blocking Reagent:

-   -   5% low fat milk in TBS

Buffer Solutions:

-   -   TBS    -   25 mM Tris/HCl buffer pH 7.5    -   +150 mM NaCl    -   TTBS    -   25 mM Tris/HCl-buffer pH 7.5    -   +150 mM NaCl    -   +0.05% Tween 20    -   PBS+0.2 mg/ml BSA    -   20 mM NaH₂PO₄ buffer pH 7.4    -   +140 mM NaCl    -   +0.2 mg/ml BSA

Antibody Solution I:

-   -   0.2 μg/ml antibody diluted in 20 ml 1% low fat milk in TBS

Antibody Solution II:

-   -   1:5000 dilution    -   Anti-Mouse-POD in 1% low fat milk in TBS

Dot Blot Procedure:

-   -   1) 1 μl each of the different Aβ-standards (in their 5 serial        dilutions) were dotted onto the nitrocellulose membrane in a        distance of approximately 1 cm from each other.    -   2) The Aβ-standards dots were allowed to dry on the        nitrocellulose membrane on air for at least 10 min at room        temperature (RT) (=dot blot)    -   3) Blocking:        -   The dot blot was incubated with 30 ml 5% low fat milk in TBS            for 1.5 h at RT.    -   4) Washing:        -   The blocking solution was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.    -   5) Antibody solution I:        -   The washing buffer was discarded and the dot blot was            incubated with antibody solution I for 2 h at RT    -   6) Washing:        -   The antibody solution I was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TBS for 10 min at RT.    -   7) Antibody solution II:        -   The washing buffer was discarded and the dot blot was            incubated with antibody solution II overnight at RT    -   8) Washing:        -   The antibody solution II was discarded and the dot blot was            incubated under shaking with 20 mil TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution was discarded and the dot blot was            incubated under shaking with 20 mil TBS for 10 min at RT.    -   9) Development:        -   The washing solution was discarded. The dot blot was            developed with 10 ml BM Blue POD Substrate for 10 min. The            development was stopped by intense washing of the dot blot            with H₂O. Quantitative evaluation was done using a            densitometric analysis (GS800 densitometer (BioRad) and            software package Quantity one, Version 4.5.0 (BioRad)) of            the dot-intensity. Only dots were evaluated that had a            relative density of greater than 20% of the relative density            of the last optically unambiguously Identified dot of the            Aβ(20-42) globulomer. This threshold value was determined            for every dot-blot independently. The calculated value            indicates the relation between recognition of Aβ(20-42)            globulomer and the respective Aβ form for the antibody given

Results are shown in FIG. 7.

Dot blot analysis of the specificity of different anti-Aβ antibodies(6E10, 5F7, 487, 10F11, 6A2, 4D10, 2F2; 3B10, 7C6, 7E5, 10C1) towardsdifferent forms of Aβ. The monoclonal antibodies tested were obtained(except for 6E10) by active immunization of mice with Aβ(20-42)globulomer, followed by selection of the fused hybridoma cells. Theindividual Aβ forms were applied in serial delusions and incubated withthe respective antibodies for immune reaction.

-   -   1. Aβ(1-42) monomer, 0.1% NH₄OH    -   2. Aβ(1-40) monomer, 0.1% NH₄OH    -   3. Aβ(1-42) monomer, 0.1% NaOH    -   4. Aβ(1-40) monomer, 0.1% NaOH    -   5. Aβ(1-42) globulomer    -   6. Aβ(12-42) globulomer    -   7. Aβ(20-42) globulomer    -   8. Aβ(1-42) fibril preparation    -   9. sAPPα (Sigma); (first dot: 1 pmol)

The anti Aβ(20-42) globulomer selective mAbs can be divided in 3 classeswith respect to the discrimination of Aβ(1-42) globulomer and Aβ(12-42)globulomer. The first class comprising the antibodies 6A2, 5F7 and 2F2recognizes preferentially Aβ(20-42) globulomer and to some extentAβ(1-42) globulomer (and also Aβ(12-42) globulomer). The second classcomprising the antibodies 10F11, 4D10 and 3810 recognizes preferentiallyAβ(20-42) globulomer and also recognizes Aβ(12-42) globulomer but to alesser extent and do not significantly recognize Aβ(1-42) globulomer.The third class comprising the antibodies 7C6, 4B7, 7E5 And 10C1recognizes Aβ(20-42) globulomer but shows no significant recognition ofthe others. All three classes do not significantly recognize monomericAβ(1-42), monomeric Aβ(1-40), A$(1-42) fibrils or sAPPα.

The selectivity profile of the anti-Aβ(20-42) globulomer antibodiesshows that the significantly elevated recognition Index in the passiveimmunization (in FIG. 6) must mainly be due to a selective recognitionof truncated Aβ(20-42) globulomer and Aβ(12-42) globulomer and to a muchlesser extent of Aβ(1-42) globulomer and not monomeric Aβ(1-42),monomeric Aβ(1-40), Aβ(1-42) fibrils or sAPPα.

Example 9 In Situ Analysis of the Specific Reaction of Aβ(20-42)Selective Antibodies to Fibrillary Aβ Peptide in the Form of Aβ Plaquesin Old TG2576 Mice and Aβ Amyloid in Meningeal Vessels

For these experiments brain material of 19 month old TG2576 mice (Hslaoet al., 1996, Science; 274(5284), 99-102) or 9 month old APP/LxPS1 mice(description as above; ReMYND, Leuven, Belgium) or autopsy material oftwo Alzheimer's disease patients (RZ16 and RZ55; obtained from BrainNet,Munich) was used. The mice overexpress human APP with the so-calledSwedish mutation (K670N/M671L; Tg2576) or with the so-called Londonmutation (V7171) in addition with the human Presenillin 1 gene with theA264E mutation (APP/LxPS1) and formed β amyloid deposits in the brainparenchyma at about 7-11 months of age and P amyloid deposits in largercerebral vessels at about 18 months of age (Tg2576). The animals weredeeply anaesthetized and transcardially perfused with 0.1 Mphosphate-buffered saline (PBS) to flush the blood. Then the brain wasremoved from the cranium and divided longitudinally. One hemisphere ofthe brain was shock-frozen, the other fixated by immersion into 4%paraformaldehyde. The immersion-fixated hemisphere was cryoprotected bysoaking in 30% sucrose in PBS and mounted on a freezing microtome. Theentire forebrain was cut into 40 μm section which were collected in PBSand used for the subsequent staining procedure. The human brain materialwas an about 1 cm³ deep-frozen block of the neocortex. A small part ofthe block was immersion-fixated in 4% paraformaldehyde and furthertreated like the mouse brain material.

Individual sections were stained with Congo Red using the followingprotocol:

Material:

-   -   Amyloid dye Congo Red kit (Sigma-Aldrich; HT-60), consisting of        alcoholic NaCl solution, NaOH solution and Congo Red solution    -   staining cuvettes    -   microscope slides SuperfrostPlus and coverslips    -   Ethanol, Xylol, embedding medium

Reagents:

-   -   NaOH diluted 1:100 with NaCl solution yields alkaline saline    -   alkaline saline diluted 1:100 with Congo Red solution yields        alkaline Congo Red solution (prepare no more than 15 min before        use, filtrate)    -   mount sections on slide and allow them to dry    -   incubate slide in staining cuvette, first for 30-40 minutes in        alkaline saline, then for 30-40 minutes in alkaline Congo Red        solution    -   rinse three times with fresh ethanol and embed over xylol

Staining was first photographed using a Zeiss Axioplan microscope andevaluated qualitatively. Red colour indicated amyloid deposits both inthe form of plaques and in larger meningeal vessels. These Results areshown in FIG. 8A. Later on, evaluation of antibody staining focused onthese structures.

Antibody staining was performed by incubating the sections with asolution containing 0.07-7.0 μg/ml of the respective antibody inaccordance with the following protocol:

Materials:

-   -   TBST washing solution (Tris Buffered Saline with Tween 20; 10x        concentrate; DakoCytomation; S3306 1:10 in Aqua bidest)    -   0.3% H₂O₂ in methanol    -   donkey serum (Serotec), 5% in TBST    -   monoclonal mouse-anti-globulomer antibody diluted in TBST    -   secondary antibody: biotinylated donkey-anti-mouse antibody        (Jackson Immuno; 715-065-150; diluted 1:500 in TBST)    -   StreptABComplex (DakoCytomation; K 0377)    -   Peroxidase Substrate Kit diaminobenzdine (=DAB; Vector        Laboratories; SK-4100)    -   SuperFrost Plus microscope slides and coverslips    -   xylol free embedding medium (Medite; X-tra Kitt)

Procedure:

-   -   transfer floating sections into ice-cold 0.3% H₂O₂ and incubate        for 30 min    -   wash for 5 min in TBST buffer    -   incubate with donkey serum I TBST for 20 minutes    -   incubate with primary antibody for 24 hours at room temperature    -   wash in TBST buffer for 5 minutes    -   Incubate with blocking serum from the Vectastain Elite ABC        peroxidase kit for 20 minutes    -   wash in TBST buffer for 5 minutes    -   incubate with secondary antibody for 60 minutes at ambient        temperature    -   wash in TBST buffer for 5 minutes    -   incubate with StreptABComplex for 60 minutes at ambient        temperature    -   wash in TBST buffer for 5 minutes    -   incubate with DAB from the Vectastain Elite ABC peroxidase kit        for 20 minutes    -   mount the section on slides, air-dry them, dehydrate them with        alcohol and embed them

Besides visual inspection of the staining, plaque staining wasadditionally quantified by graphically excising 10 randomly selectedplaques from the histological images using the ImagePro 5.0 Imageanalysis system and determining their average greyscale value. Opticaldensity values were calculated from the greyscale values by subtractingthe mean background density of the stained material from the density ofamyloid plaques (0%—no plaque staining above surrounding background,100%—no transmission/maximal staining), and the differences betweencontrol and antibodies and between 6G1 and the antibodies selective forAβ(20-42), respectively, were tested for statistical significance withANOVA.

Results of the staining in Tg2576 and APP/LxPS1 mice are shown in FIG. 8B-D and H.

Binding of different antibodies at a concentration of 0.7 μg/ml intransversal section of the neocortices of transgenic TG 2576 mice at 19months of age:

-   -   C) Parenchymal Aβ deposits (amyloid plaques) were stained only        with 6G1 and 6E10 but not with the globulomer selective        antibodies (i.e. 5F7, 2F2, 6A2, 4010, 10F11, 3B10, 7C6, 7E5 and        10C1)    -   D) All globulomer selective antibodies (i.e. 5F7, 2F2, 6A2,        4D10, 10F11, 3B10, 7C6, 7E5 and 10C1) showed significantly less        parenchymal plaque staining compared to the commercially        available antibodies 6E10 and 4G8.

Binding of different antibodies at a concentration of 0.07-7.0 μg/ml intransversal section of the neocortices of transgenic APP/LxPS1 mice at11 months of age:

-   -   E) Parenchymal Aβdeposits (amyloid plaques) were significantly        more and at lower concentrations stained with 6G1, 6E10 and 4G8        than with the globulomer selective antibodies (i.e. 5F7, 2F2,        6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

All amyloid deposits had been verified by congophilic staining before(Congo Red; see FIG. 8A). Bar=100 μm.

Evaluation of brown DAB deposits showed that the Ap-unselective 6G1 and6E10 antibodies stained plaques and meningeal vessels, whereas theAβ(20-42) globulomer selective antibodies 5F7, 2F2, 6A2, 4D10, 10F11,3B10, 7C6, 7E5 and 10C1 did not. This finding demonstrates that there isno or markedly less binding of these antibodies to Aβ fibrils or otherAβ species present in the amyloid structures in vivo. This reducedbinding is supposed to reduce the danger of side effects induced by toofast dissolution of plaques and subsequent increase in soluble Aβ orneuroinflammation due to the interaction of plaque-bound antibodies withmicroglia.

Results of the staining in human Alzheimer's disease brain are shown inFIG. 8 B, F-H.

Binding of different antibodies at a concentration of 0.7 μg/ml intransversal section of the neocortex of patient RZ55:

-   -   B) Parenchymal Aβ deposits (amyloid plaques) were stained only        with 6G1 and 6E10 but not with the globulomer selective        antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3810, 7C6, 7E5 and        10C1).    -   F) All globulomer selective antibodies (i.e. 5F7, 2F2, 6A2,        4D10, 10F11, 3810, 7C6, 7E5 and 10C1) showed significantly less        staining compared to the commercially available antibodies 6E10        and 4G8.    -   H) Vascular Aβ deposits (arrows) were stained only with 6G1 and        6E10 but not with globulomer selective antibodies (i.e. 5F7,        2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

Binding of different antibodies at a concentration of 0.07-7.0 μg/ml intransversal section of the neocortices of transgenic APP/LxPS1 mice at11 months of age:

-   -   G) Parenchymal Aβ deposits (amyloid plaques) were significantly        more and at lower concentrations stained with 6G1, 6E10 and 4G8        than with the globulomer selective antibodies (i.e. 5F7, 2F2,        6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

All amyloid deposits had been verified by congophilic staining before(Congo Red; see FIG. 8A)

Evaluation of brown DAB deposits showed that the Aβ-unselective 6G1 and6E10 antibodies stained plaques and meningeal vessels, whereas theAβ(20-42) globulomer selective antibodies 5F7, 2F2, 6A2, 4010, 10F11,3B10, 7C6, 7E5 and 10C1 did not. Commercially available antibodies 6E10and 4G8 showed stronger staining compared to globulomer selectiveantibodies, but less staining than 6G1. This finding confirms thestaining pattern in APP transgenic mice where there is no or markedlyless binding of the globulomer selective antibodies to AP fibrils orother Aβ species present in the amyloid structures in vivo. This reducedbinding to human amyloid is supposed to reduce the danger of sideeffects induced by too fast dissolution of plaques and subsequentincrease in soluble Aβ or neuroinflammation due to the interaction ofplaque-bound antibodies with microglia.

Example 10 Anti-AP-Antibody Titer and Dot-Blot Selectivity Profile inPlasma of TG2576 Mice Approximately One Year after Active Immunization

Approximately one year after the last immunization (with Aβ(20-42)globulomer, Aβ(12-42) globulomer, Aβ(1-42) monomer and vehicle) of Tg2576 mice (from example 9) plasma samples were assessed for anti-A,antibodies produced and still present. To this end, dilution series ofthe different forms of Aβ(1-42) in the concentration range from 100pmol/μl to 0.01 pmol/μl in PBS+0.2 mg/ml BSA were made. Of each sample,1 μl was applied to a nitrocellulose membrane. Detection was performedwith suitable mouse plasma samples (diluted 1:400). Staining was donewith anti-mouse-lgG conjugated alkaline phosphatase and addition of thestaining reagent NBT/BCIP

Aβ-Standards for Dot-Blot 1. Aβ(1-42) Globulomer

The preparation of the Aβ(1-42) globulomer is described in example 1a.

2. HFIP Pretreated Aβ(1-42) Monomer in Pluronic F68

3 mg Aβ(1-42), (Bachem Inc.; cat. no. H-1368) were dissolved in 0.5 mlHFIP (6 mg/ml suspension) in an 1.7 ml Eppendorff tube and was shaken(Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37° C. till a clearsolution was obtained. The sample was dried in a SpeedVac concentrator(1.5 h) and resuspended in 13.2 μl DMSO, shaken for 10 sec., followed bysonification (20 sec), and shaking (e.g. in Eppendorff Thermo mixer,1400 rpm) for 10 min. 6 ml 20 mM NaH₂PO₄; 140 mM NaCl; 0.1% PluronicF68; pH 7.4 was added and stirred for 1 h at room temperature. Thesample was centrifuged for 20 min at 3000 g. The supernatant wasdiscarded and the precipitate solved in 0.6 ml 20 mM NaH₂PO₄; 140 mMNaCl; 1% Pluronic F68, pH 7.4 3.4 ml H₂O was added and stirred for 1 hat room temperature followed by 20 min centrifugation at 3000 g. Eightaliquots of each 0.5 ml of the supernatant were stored at −20° forfurther use.

3. Aβ(20-42) Globulomer

The preparation of the Aβ(20-42) globulomer is described in example 1c.

4. Aβ(12-42) Globulomer

The preparation of the Aβ(1-42) globulomer is described in example 1d.

5. Aβ(1-40) Monomer, HFIP Pretreated, 5 mM in DMSO

1 mg Aβ(1-40), (Bachem Inc, cat. no. H-1194) were suspended in 0.25 mlHFIP (4 mg/ml suspension) in an Eppendorff tube. The tube was shaken(e.g. In Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37° C. to get aclear solution and afterwards dried in a SpeedVac concentrator (for 1.5h). The sample was redissolved in 46 μl DMSO (21.7 mg/ml solution=5 mM),shaken for 10 sec and subsequently sonicated for 20 sec. After shaking(e.g. In Eppendorff Thermo mixer, 1400 rpm) for 10 min the sample isstored at −20° C. for further use.

6. Aβ(1-42) monomer, 0.1% NH₄OH

1 mg Aβ(1-42) (Bachem Inc., cat no. H-1368) were dissolved in 0.5 ml0.1% NH₄OH in H₂O (freshly prepared) (=2 mg/ml) and immediately shakenfor 30 sec. at room temperature to get a clear solution. The sample wasstored at −20° C. for further use.

7. Aβ(1-42) Fibrils

1 mg Aβ(1-42) (Bachem Inc. Catalog Nr.: H-1368) were solved in 500 μlaqueous 0.1% NH₄OH (Eppendorff tube) and the sample was stirred for 1min at room temperature. 100 μl of this freshly prepared Aβ(1-42)solution were neutralized with 300 μl 20 mM NaH₂PO₄; 140 mM NaCl, pH7.4. The pH was adjusted to pH 7.4 with 1% HCl. The sample was incubatedfor 24 h at 37° C. and centrifuged (10 min at 10000 g). The supernatantwas discarded and the fibril pellet resuspended with 400 μl 20 mMNaH₂PO₄; 140 mM NaCl, pH 7.4 by vortexing for 1 min.

8. sAPPα

Supplied from Sigma (cat.no. S9564; 25 μg in 20 mM NaH₂PO₄; 140 mM NaCl;pH 7.4). The sAPPα was diluted with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4,0.2 mg/ml BSA to 0.1 mg/ml (=1 pmol/μl).

Materials for Dot Blot: Aβ-Standards:

-   -   Serial dilution of Aβ-antigens in 20 mM NaH₂PO₄, 140 mM NaCl, pH        7.4+0.2 mg/ml BSA        -   1) 100 pmol/μl        -   2) 10 pmol/μl        -   3) 1 pmol/μl        -   4) 0.1 pmol/μl        -   5) 0.01 pmol/μl

Nitrocellulose:

-   -   Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45        μm); BIO-RAD

Anti-Mouse-AP:

-   -   AQ330A (Chemicon)

Detection Reagent:

-   -   NBT/BCIP Tablets (Roche)

Bovine Serum Albumin, (BSA):

-   -   A-7888 (Fa. SIGMA)

Blocking Reagent:

-   -   5% low fat milk in TBS

Buffer Solutions:

-   -   TBS    -   25 mM Tris/HCl-buffer pH 7.5    -   +150 mM NaCl    -   TTBS    -   25 mM Tris/HCl-buffer pH 7.5    -   +150 mM NaCl    -   +0.05% Tween 20    -   PBS+0.2 mg/ml BSA    -   20 mM NaH₂PO₄ buffer pH 7.4    -   +140 mM NaCl    -   +0.2 mg/ml BSA

Antibody Solution I:

-   -   Plasma of the TG2576 mice actively immunized 1/400 diluted in 20        ml 1% low fat milk in TBS

Antibody Solution II:

-   -   1:5000 dilution    -   Anti-Mouse-AP in 1% low fat milk in TBS

Dot Blot Procedure:

-   -   1) 1 μl each of the different Aβ standards (in their 5 serial        dilutions) were dotted onto the nitrocellulose membrane in        approximately 1 cm distance from each other.    -   2) The Aβ standards dots are allowed to dry on the        nitrocellulose membrane on air for at least 10 min at room        temperature (RT) (=dot blot)    -   3) Blocking:        -   The dot blot is incubated with 30 ml 5% low fat milk in TBS            for 1.5 h at RT.    -   4) Washing:        -   The blocking solution is discarded and the dot blot            incubated under shaking with 20 ml TTBS for 10 min at RT.    -   5) Antibody solution 1:        -   The washing buffer is discarded and the dot blot incubated            with antibody solution I overnight at RT    -   6) Washing:        -   The antibody solution I is discarded and the dot blot            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution is discarded and the dot blot incubated            under shaking with 20 ml TTBS for 10 min at RT. The washing            solution is discarded and the dot blot incubated under            shaking with 20 ml TBS for 10 min at RT.    -   7) Antibody solution II:        -   The washing buffer is discarded and the dot blot incubated            with antibody solution II for 1 h at RT.    -   8) Washing:        -   The antibody solution II is discarded and the dot blot            incubated under shaking with 20 ml TTBS for 10 min at RT.            The washing solution is discarded and the dot blot incubated            under shaking with 20 ml TTBS for 10 min at RT. The washing            solution is discarded and the dot blot incubated under            shaking with 20 ml TBS for 10 min at RT.    -   9) Development:        -   The washing solution is discarded. 1 tablet NBT/BCIP is            dissolved in 20 ml H₂O and the dot blot is incubated for 5            min with this solution. The development is stopped by            intensive washing with H₂O.

Results are shown in FIG. 9.

Sera of different immunization groups: a) Aβ(20-42) globulomers; b)Aβ(12-42) globulomers; c) Aβ(1-42) monomer, 0.1% NH₄OH; d) vehiclecontrol were tested against different Aβ forms in a dot blot fordiffering antibody profiles.

1. Aβ(1-42) globulomer

2. Aβ(1-42) monomer, HFIP pretreated, in 0.1% Pluronic F68

3. Aβ(20-42) globulomer

4. Aβ(12-42) globulomer

5. Aβ(1-40) monomer, HFIP pretreated, 5 mM in DMSO

6. Aβ(1-42) monomer, dissolved in 0.1% NH₄OH

7. Aβ(1-42) fibril preparation

8. sAPPα (Sigma); (first dot: 1 pmol)

In contrast to the active immunizations with either vehicle as controlor Aβ(1-42) monomer the immunization with Aβ(20-42) globulomer orAβ(12-42) globulomer exhibits even after approximately one year of thelast immunization a high titer of antibodies. These antibodies areselective for the Aβ(20-42) globulomer in the case of the Aβ(20-42)globulomer immunization or Aβ(20-42) globulomer and Aβ(12-42) globulomerselective in the case of the Aβ(12-42) globulomer immunization. Thisshows that the truncated Aβ(20-42) globulomer and Aβ(12-42) globulomerrepresent a very good antigen and that antibodies directed against thempersist very long in vivo.

(Note that on some dot-blots an unspecific staining signal is observedwhich is most likely a cross reaction of murine antibodies to the BSAused in the serial dilutions of the Aβ peptides.)

Example 11 Brain-Levels of Aβ(20-42) Globulomer Epitopes in Alzheimer'sDisease Patients SDS-DTT-Brain Extract:

AD brain samples: RZ 16; RZ 52 und RZ 55 (obtained from Brain Net,Munich) Control sample: RZ 92 (obtained from Brain Net, Munich)

One tablet of Complete Protease Inhibitor (Roche, Cat. No. 1697 498) isdissolved in 1 ml H₂O (=protease inhibitor solution). 100 mg of AD brainsample are homogenized in 2.5 ml NaH₂PO₄, 140 mM NaCl, 0.05% Tween 20,0.5% BSA (supplemented with 25 μl protease inhibitor solution) with 20strokes in a glass potter. The suspension is sonified for 30 sec on ice,then incubated at 37° C. for 16 h. The suspension is centrifuged at100,000 g and 8° C. for one hour, then the supernatant is collected. Theresidue is dissolved in 5 mM NaH₂PO₄, 35 mM NaCl, pH 7.4 and homogenizedwith 10 strokes in a glass potter. 75 μl of 10% SDS and 125 μl of 0.16mg/ml DTT are added and stirred for 20 minutes at ambient temperature.The sample was centrifuged for 10 minutes at 10,000 g, and thesupernatant is stored overnight at −20° C. Before use the supernatant isthawed and centrifuged for another 10 min at 10,000 g. The supernatant(=SDS/DTT brain extract) is used for ELISA.

a) Sandwich-ELISA for Aβ(20-42) Globulomer Epitope Reagent List:

-   -   1. F96 Cert. Maxisorp NUNC-Immuno Plate (Cat.No.:439454)    -   2. Binding antibody: 5F7, 7C6, 10F11    -   3. Coupling buffer:        -   100 mM sodium hydrogen carbonate, pH9.6    -   4. Blocking reagent for ELISA (Roche Diagnostics GmbH Cat.No.:        1112589)    -   5. PBST buffer:        -   20 mM NaH₂PO₄, 140 mM NaCl, 0.05% Tween 20, pH 7.4    -   6. Aβ(20-42) calibration standard    -   7. Primary antibody:        -   anti-Aβ pRAb BA199; affinity purified (by Aβ(1-42)            globulomer-Sepharose) IgG solution in PBS; Konz.: 0.22 mg/ml    -   8. Secondary antibody:        -   anti-rabbit-POD conjugate; (Jackson ImmunoResearch, Cat.No.:            111-036-045)    -   9. Development:        -   TMB; (Roche Diagnostics GmbH Cat.No.: 92817060) 42 mM in            DMSO        -   3% H₂O₂ in H₂O    -   100 mM sodium acetate, pH4.9    -   Stop solution: 2M sulfuric acid

Preparation of Reagents:

-   -   1. Binding Antibody:        -   The individual binding antibodies 5F7, 7C6 and 10F11 are            diluted to a final concentration of 0.7 μg/ml in coupling            buffer.    -   2. Blocking reagent        -   For preparation of the blocking stock solution the blocking            reagent is dissolved in 100 ml H₂O and stored at −20° C. in            aliquots of 10 ml each.        -   3 ml of the blocking stock solution are diluted with 27 ml            H₂O for blocking one ELISA plate.    -   3. Aβ(20-42) calibration standard (CS 1)

The preparation of the Aβ(1-42) globulomer is described in example 1a.

The Aβ(20-42) globulomer protein concentration was determined (6.81mg/ml) after Bradford (BioRad). 14.68 μl Aβ(20-42) globulomer (6.81mg/ml) are diluted in 10 ml 20 mM NaH₂PO₄, 140 mM NaCl, 0.05% Tween20,pH 7.4, 0.5% BSA (=10 μg/ml). 10 μl of the 10 μg/ml solution are furtherdiluted in 10 ml 20 mM NaH₂PO₄, 140 mM NaCl, 0.05% Tween20, pH 7.4, 0.5%BSA (=10 ng/ml=CS1)

Calibration Standards for Aβ(20-42):

final volume of concentration Calibration calibration Aβ(20-42) standardstandard PBST + 0.5% BSA (pg/ml) CS1.1    1 ml of CS1    0 ml 10000CS1.2 0.316 ml of CS1.1 0.684 ml 3160 CS1.3 0.316 ml of CS1.2 0.684 ml1000 CS1.4 0.316 ml of CS1.3 0.684 ml 316 CS1.5 0.316 ml of CS1.4 0.684ml 100 CS1.6 0.316 ml of CS1.5 0.684 ml 31.6 CS1.7 0.316 ml of CS1.60.684 ml 10 CS1.8 0.0  1.0 ml 0.0

SDS/DTT-Brain Extracts:

SDS/DTT-brain extracts=E#

(#represents the 4 human brain samples (1) RZ 16; (2) RZ 52; (3) RZ 55;(4) RZ 92)

extraction PBST + 0.5% dilution sample volume of extraction sample BSAfactor E#. 1    1 ml of E#  0.0 ml direct E#. 2 0.316 ml of E#. 1 0.684ml 1:3.16 E#. 3 0.316 ml of E#. 1 0.684 ml 1:10 E#. 4 0.316 ml of E#. 10.684 ml 1:31.6

-   -   4. Primary Antibody:        -   The anti-Aβ pRAb stock solution is diluted to 0.05 μg/ml in            PBST+0.5% BSA. The antibody solution is used immediately.    -   5. Secondary Antibody:        -   Lyophilized anti-rabbit-POD conjugate is dissolved in 0.5 ml            H₂O and mixed with 500 μl glycerol. The antibody concentrate            is then stored at −20° C. in aliquots of 100 μl. The            concentrate is diluted 1:10,000 in PBST buffer. The antibody            solution is used immediately.    -   6. TMB solution:        -   20 ml of 100 mM sodium acetate, pH 4.9, are mixed with 200            μl TMB solution and 29.5 μl of 3% hydrogen peroxide. This            solution is used immediately.        -   ELISA-Plate for Aβ(20-42):        -   Calibration standards (CS1.1-CS1.8) and SDS/DTT-brain            extracts of the 4 human brain samples (1) RZ 16; (2) RZ            52; (3) RZ 55; (4) RZ 92 (=E1-E4 in their 4 serial dilutions            E#.1-E#.4) are determined in double:

1 2 3 4 5 6 7 8 9 10 11 12 A CS1.1 CS1.1 E1.1 E1.1 E3.1 E3.1 B CS1.2CS1.2 E1.2 E1.2 E3.2 E3.2 C CS1.3 CS1.3 E1.3 E1.3 E3.3 E3.3 D CS1.4CS1.4 E1.4 E1.4 E3.4 E3.4 E CS1.5 CS1.5 E2.1 E2.1 E4.1 E4.1 F CS1.6CS1.6 E2.2 E2.2 E4.2 E4.2 G CS1.7 CS1.7 E2.3 E2.3 E4.3 E4.3 H CS1.8CS1.8 E2.4 E2.4 E4.4 E4.4

-   -   -   ELISA is performed with each of the binding monoclonal            antibodies 5F7, 7C6, 10F11.

Procedure

-   -   1. Add 100 μl mAb solution per well. Incubate the ELISA plate        overnight at +6° C. (fridge).    -   2. Decant the antibody solution and wash wells three times with        250 μl PBST buffer each.    -   3. Add 250 μl/well of blocking solution. Incubate for 2 hours at        ambient temperature.    -   4. Decant the blocking solution and wash wells three times with        250 μl PBST buffer each.    -   5. Add 100 μl/well each of calibration standards and SDS/DTT        brain extracts. Incubate plate for 2 hours at ambient        temperature, then overnight at 6° C.    -   6. Decant the calibration standards and SDS/DTT brain extracts        solution and wash wells three times with 250 μl PBST buffer        each.    -   7. Add 200 μl/well of primary antibody solution and incubate for        1 hour at ambient temperature.    -   8. Decant the primary antibody solution and wash wells three        times with 250 μl PBST buffer each.    -   9. Add 200 μl/well of secondary antibody solution and incubate        for 1 hour at ambient temperature.    -   10. Decant the secondary antibody solution and wash wells three        times with 250 μl PBST buffer each.    -   11. Add 100 μl/well of TMB solution.    -   12. Monitor plate colour during development (5-15 min at ambient        temperature) and terminate reaction by adding 50 μl/well of stop        solution when an appropriate colour has developed.    -   13. Measure extinction at 450 nm.    -   14. Calculate results using calibration.    -   15. Evaluation: If the extinctions of the samples are beyond the        linear calibration range, dilute them again and repeat.

Results are shown in FIG. 10.

Brain levels of Aβ(20-42) globulomer epitopes in brain extracts from ADpatients and control subjects

A sandwich ELISA was used to assess brain extracts for their truncatedAβ(20-42) globulomer epitope content. ELISAs with the respectiveantibodies against the Aβ(20-42) globulomer were used for calibration.

Extraction of Alzheimer's disease brain tissue shows that the Aβ(20-42)globulomer epitope content is significantly elevated compared to acontrol patient. This shows that Indeed the Aβ(20-42) globulomer epitopeis a relevant Au-species in human Alzheimer's disease brain and not onlyrelevant for Alzheimer's disease animal models. Antibodies directedagainst the Aβ(20-42) globulomer epitope therefore are highly desirablefor the treatment of Alzheimer's disease.

Example 12 Development of Anti-Aβ(20-42) Globulomer Hybridoma Cell Lines

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references Incorporatedherein by reference in their entireties). The term “monoclonal antibody”as used herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

The particular protocol used to produce the antibodies described hereinis as follows:

Immunization of mice: Balb/c and A/J mice (6-8 week old) were immunizedsubcutaneously with 50 ug of antigen in CFA. Animals were boosted everythree weeks with 50 ug of antigen in Immuneasy™ (Qiagen) for a total ofthree boosts. Four days prior to fusion, mice were boosted with 10 ug ofantigen Intravenously.

Cell fusion and hybridoma screening: Spleen cells from Immunized animalswere fused with SP2/0-Ag14 myeloma cells at a ratio of 5:1 usingstandard techniques. Seven to ten days post fusion, when macroscopiccolonies were observed, SN were tested by ELISA for antibody toAβ(20-42) globulomer Cells from ELISA positive wells were scaled up andcloned by limiting dilution.

Antibody isotype determination: The isotype of the anti-Aβ(20-42)globulomer mAbs was determined using the Zymed EIA isotyping kit.

Scale up and purification of monoclonal antibodies: Hybridomas wereexpanded into media containing 5% Low IgG. Fetal bovine serum (Hyclone).Supernatant was harvested and concentrated. mAb was purified usingProtein A chromatography and dialyzed into PBS.

Serum titers: Ten mice were immunized with the Aβ(20-42) globulomer. Allmice seroconverted with ELISA titers (½ Max OD 450 nm) of 1:5000-10,000.

Designations of Hybridomas Producing Monoclonal Antibodies

Internal designations of Abbott Laboratories used for the deposits.

Deposited Cell Lines:

1) ML 13-7C6.1D4.4A9.5G8 (also referred to herein as “7C6”)

2) ML15-5F7.5B10 (also referred to herein as “5F7”)

3) ML15-10F11.3D9 (also referred to herein as “10F11”)

4) ML15-4B7.3A6 (also referred to herein as “4B7”)

5) ML15-2F2.3E12 (also referred to herein as “2F2”)

6) ML15-6A2.4B10 (also referred to herein as “6A2”)

7) ML13-4D10.3F3 (also referred to herein as “4D10”)

8) ML15-7E5.5E12 (also referred to herein as “7E5”)

9) ML15-10C1.5C6.3H4 (also referred to herein as “10C1”)

10) ML15-3B10.2D5.3F1 (also referred to herein as “3B10”)

1.-116. (canceled)
 117. An antibody selected from the group consistingof an isolated antibody, a monoclonal antibody and a recombinantantibody, wherein said antibody has a binding affinity to an Aβ(20-42)globulomer that is greater than the binding affinity of the antibody toan Aβ(1-42) globulomer, wherein the binding affinity of the antibody tothe Aβ(20-42) globulomer is at least 10 times greater than the bindingaffinity of the antibody to the Aβ(1-42) globulomer, wherein theantibody is humanized and binds to the same epitope as a monoclonalantibody 7C6 obtainable from a hybridoma designated by American TypeCulture Collection deposit number PTA-7240.
 118. The antibody of claim117, wherein the antibody comprises at least 2 variable domain CDR sets.119. The antibody of claim 118, wherein the at least 2 variable domainCDR sets are VH 7C6 CDR Set and VL 7C6 CDR Set.
 120. An antibodyselected from the group consisting of an isolated antibody, a monoclonalantibody and a recombinant antibody, wherein said antibody comprises twovariable domains, wherein said two variable domains are SEQ ID NO:11 andSEQ ID NO:12.
 121. The antibody of claim 120, wherein the antibodycomprises a constant region.
 122. The antibody of claim 121, wherein theantibody comprises a heavy chain constant region selected from the groupconsisting of IgG, IgG2, IgG3, IgG4, IgM, IgA, IgD and IgE constantregions.
 123. The antibody of claim 122, wherein the antibody comprisesan IgG1 heavy chain constant region.
 124. The antibody of claim 122,wherein the constant region is a human constant region.
 125. Theantibody of claim 121, wherein the constant region comprises the aminoacid sequence of SEQ ID NO:39.
 126. The antibody of claim 125, whereinthe antibody possesses a human glycosylation pattern.
 127. Monoclonalantibody (7C6) obtainable from a hybridoma designated by American TypeCulture Collection deposit number PTA-7240.
 128. Antigen-binding moietyof an antibody of claim
 117. 129. The antigen-binding moiety of claim128, wherein the moiety is selected from the group consisting of a Fabfragment, a F(ab′)₂ fragment and a single chain Fv fragment of theantibody.
 130. Hybridoma designated by an American Type CultureCollection deposit number PTA-7240.
 131. A composition comprising saidantibody of claim 117 or said antigen-binding moiety of claim
 128. 132.The composition of claim 131, wherein said composition is apharmaceutical composition and further comprises a pharmaceuticalacceptable carrier.